Methods and compositions for protein sequencing

Abstract

To provide methods for sequencing polypeptides.SOLUTION: Provided is a method including: obtaining data during a degradation process of a polypeptide; analyzing the data to determine portions of the data corresponding to amino acids that are sequentially exposed at a terminus of the polypeptide during the degradation process; and outputting an amino acid sequence representative of the polypeptide. The data may be indicative of amino acid identity at the terminus of the polypeptide during the degradation process.SELECTED DRAWING: Figure 1A

Description

[Technical field]

[0001] The present invention relates to methods and compositions for protein sequencing. [Background technology]

[0002] Proteomics has emerged as an important and necessary complement to genomics and transcriptomics in the study of biological systems. Proteomic analysis of individual organisms can provide insight into cellular processes and reaction patterns that can lead to improved diagnostic and therapeutic strategies. The complexity surrounding protein structure, composition, and modifications presents challenges in determining large-scale protein sequencing information for biological samples. Summary of the Invention [Means for solving the problem]

[0003] In some aspects, the present application provides methods and compositions for determining amino acid sequence information from a polypeptide (e.g., for sequencing one or more polypeptides). In some embodiments, amino acid sequence information can be determined for a single polypeptide molecule. In some embodiments, for example, for a single polypeptide molecule, the relative positions of two or more amino acids of the polypeptide are determined. In some embodiments, one or more amino acids of the polypeptide are labeled (e.g., directly or indirectly) and the relative positions of the labeled amino acids of the polypeptide are determined.

[0004] In some aspects, the present application provides a method comprising obtaining data during a degradation process of a polypeptide. In some embodiments, the method further comprises analyzing the data to determine portions of the data that correspond to amino acids that are sequentially exposed at a terminus of the polypeptide during the degradation process. In some embodiments, the method further comprises outputting an amino acid sequence corresponding to the polypeptide. In some embodiments, the data is indicative of amino acid identity at the termini of the polypeptide during the degradation process. In some embodiments, the data is indicative of signals generated by one or more amino acid recognition molecules that bind to different types of terminal amino acids at the termini during the degradation process. In some embodiments, the data is indicative of luminescent signals generated during the degradation process. In some embodiments, the data is indicative of electrical signals generated during the degradation process.

[0005] In some embodiments, analyzing the data further comprises detecting a series of cleavage events and determining a portion of the data between successive cleavage events. In some embodiments, analyzing the data further comprises determining a type of amino acid for each of the distinct portions. In some embodiments, each of the distinct portions includes a pulse pattern (e.g., a characteristic pattern), and analyzing the data further comprises determining a type of amino acid for the one or more portions based on its respective pulse pattern. In some embodiments, determining the type of amino acid further comprises identifying an amount of time within the portion when the data is above a threshold and comparing the amount of time and duration for the portion. In some embodiments, determining the type of amino acid further comprises identifying at least one pulse duration for each of the one or more portions. In some embodiments, determining the type of amino acid further comprises identifying at least one inter-pulse duration for each of the one or more portions. In some embodiments, the amino acid sequence comprises a series of amino acids corresponding to the portions.

[0006] In some aspects, the present application provides a system including at least one hardware processor and at least one non-transitory computer readable storage medium storing processor executable instructions that, when executed by the at least one hardware processor, cause the at least one hardware processor to perform a method according to the present application. In some aspects, the present application provides at least one non-transitory computer readable storage medium storing processor executable instructions that, when executed by the at least one hardware processor, cause the at least one hardware processor to perform a method according to the present application.

[0007] In some aspects, the present application provides a method for sequencing a polypeptide. In some embodiments, the method comprises contacting a single polypeptide molecule with one or more terminal amino acid recognition molecules. In some embodiments, the method further comprises obtaining sequence information about the single polypeptide molecule by detecting a series of signal pulses indicative of the association of the one or more terminal amino acid recognition molecules with successive amino acids exposed at the termini of the single polypeptide molecule during degradation. In some embodiments, the sequence of most or all of the amino acids of the single polypeptide molecule is determined. In some embodiments, the series of signal pulses is a series of real-time signal pulses.

[0008] In some embodiments, association of one or more terminal amino acid recognition molecules with each type of terminally exposed amino acid produces a characteristic pattern in the series of signal pulses that is distinct from other types of terminally exposed amino acids. In some embodiments, the characteristic pattern of signal pulses corresponds to individual association events between terminal amino acid recognition molecules and terminally exposed amino acids. In some embodiments, the characteristic pattern corresponds to a series of reversible terminal amino acid recognition molecule binding interactions with terminally exposed amino acids of a single polypeptide molecule. In some embodiments, the characteristic pattern is indicative of terminally exposed amino acids and adjacent amino acids (e.g., amino acids of the same type or different types) of a single polypeptide molecule.

[0009] In some embodiments, the single polypeptide molecule is degraded with a cleavage reagent that removes one or more amino acids from the terminus of the single polypeptide molecule. In some embodiments, the method further comprises detecting a signal indicative of association of the cleavage reagent with the terminus. In some embodiments, the cleavage reagent comprises a detectable label (e.g., a luminescent label, a conductivity label). In some embodiments, the single polypeptide molecule is immobilized on a surface. In some embodiments, the single polypeptide molecule is immobilized on a surface via a terminus distal to the terminus with which the one or more terminal amino acid recognition molecules are associated. In some embodiments, the single polypeptide molecule is immobilized on a surface via a linker (e.g., a solubilizing linker that includes a biomolecule).

[0010] In some aspects, the present application provides a method for sequencing a polypeptide, comprising contacting a single polypeptide molecule in a reaction mixture with a composition comprising one or more terminal amino acid recognition molecules and a cleavage reagent. In some embodiments, the method further comprises detecting a series of signal pulses indicative of the association of one or more terminal amino acid recognition molecules with the terminus of the single polypeptide molecule in the presence of the cleavage reagent. In some embodiments, the series of signal pulses indicative of the series of amino acids exposed at the terminus over time as a result of the cleavage of the terminal amino acids by the cleavage reagent.

[0011] In some aspects, the present application provides a method of sequencing a polypeptide comprising: (a) identifying a first amino acid at a terminus of the single polypeptide molecule; (b) removing the first amino acid to expose a second amino acid at the terminus of the single polypeptide molecule; and (c) identifying the second amino acid at the terminus of the single polypeptide molecule. In some embodiments, (a)-(c) are performed in a single reaction mixture. In some embodiments, (a)-(c) are performed sequentially. In some embodiments, (c) is performed prior to (a) and (b). In some embodiments, the single reaction mixture comprises one or more terminal amino acid recognition molecules. In some embodiments, the single reaction mixture comprises a cleavage reagent. In some embodiments, the first amino acid is removed by the cleavage reagent. In some embodiments, the method further comprises determining a sequence (e.g., a partial sequence or a complete sequence) of the single polypeptide molecule by repeating the steps of removing and identifying one or more amino acids at the terminus of the single polypeptide molecule.

[0012] In some aspects, the present application provides a method for identifying amino acids of a polypeptide, comprising contacting a single polypeptide molecule with one or more amino acid recognition molecules that bind to the single polypeptide molecule.In some embodiments, the method further comprises detecting a series of signal pulses indicative of the association of the one or more amino acid recognition molecules with the single polypeptide molecule under polypeptide degradation conditions.In some embodiments, the method further comprises identifying a first type of amino acid of the single polypeptide molecule based on a first characteristic pattern of the series of signal pulses.

[0013] In some aspects, the present application provides methods for identifying terminal amino acids (e.g., N-terminal or C-terminal amino acids) of a polypeptide. In some embodiments, the methods include contacting the polypeptide with one or more labeled affinity reagents (e.g., one or more amino acid recognition molecules) that selectively bind to one or more types of terminal amino acids at the termini of the polypeptide. In some embodiments, the methods further include identifying the terminal amino acids at the termini of the polypeptide by detecting an interaction between the polypeptide and the one or more labeled affinity reagents.

[0014] In yet another aspect, the present application provides a method for sequencing a polypeptide by Edman-type degradation reaction. In some embodiments, the Edman-type degradation reaction can be performed by contacting the polypeptide with various reaction mixtures for the purpose of either detection or cleavage (e.g., compared to a kinetic sequencing reaction that can include detection and cleavage using a single reaction mixture).

[0015] Thus, in some aspects, the present application provides a method for determining the amino acid sequence of a polypeptide comprising: (i) contacting the polypeptide with one or more labeled affinity reagents that selectively bind to one or more types of terminal amino acids at a terminus of the polypeptide. In some embodiments, the method further comprises (ii) identifying a terminal amino acid (e.g., an N-terminal or C-terminal amino acid) at a terminus of the polypeptide by detecting an interaction of the polypeptide with the one or more labeled affinity reagents. In some embodiments, the method further comprises (iii) removing the terminal amino acid. In some embodiments, the method further comprises (iv) repeating (i)-(iii) one or more times at the terminus of the polypeptide to determine the amino acid sequence of the polypeptide.

[0016] In some embodiments, the method further comprises, after (i) and before (ii), removing any one or more labeled affinity reagents that do not selectively bind to the terminal amino acid. In some embodiments, the method further comprises, after (ii) and before (iii), removing any one or more labeled affinity reagents that selectively bind to the terminal amino acid.

[0017] In some embodiments, removing the terminal amino acid (e.g., (iii)) comprises modifying the terminal amino acid by contacting the terminal amino acid with an isothiocyanate (e.g., phenylisothiocyanate) and contacting the modified terminal amino acid with a protease that specifically binds to and removes the modified terminal amino acid. In some embodiments, cleaving the terminal amino acid (e.g., (iii)) comprises modifying the terminal amino acid by contacting the terminal amino acid with an isothiocyanate and subjecting the modified terminal amino acid to acidic or basic conditions sufficient to remove the modified terminal amino acid.

[0018] In some embodiments, identifying the terminal amino acid comprises identifying the terminal amino acid as being one type of one or more types of terminal amino acids to which the one or more labeled affinity reagents bind, In some embodiments, identifying the terminal amino acid comprises identifying the terminal amino acid as being a type other than the one or more types of terminal amino acids to which the one or more labeled affinity reagents bind.

[0019] In some aspects, the present application provides amino acid recognition molecules that include a shielding element, for example for enhanced photostability during polypeptide sequencing reactions. In some aspects, the present application provides an amino acid recognition molecule having formula (I): A-(Y) n -D (I) wherein A is an amino acid binding moiety comprising at least one amino acid recognition molecule; each of the Y's is a polymer forming a covalent or non-covalent linking group; n is an integer from 1 to 10, inclusive; and D is a labeling moiety comprising at least one detectable label. In some embodiments, D is less than 200 Å in diameter. In some embodiments, -(Y) n - is at least 2 nm in length (e.g., at least 5 nm, at least 10 nm, at least 20 nm, at least 30 nm, at least 50 nm, or more in length). In some embodiments, -(Y) n - is about 2 nm to about 200 nm in length (e.g., about 2 nm to about 100 nm, about 5 nm to about 50 nm, or about 10 nm to about 100 nm in length). In some embodiments, each Y is independently a biomolecule or a dendritic polymer (e.g., a polyol, a dendrimer). In some embodiments, the present application provides a composition comprising an amino acid recognition molecule of formula (I). In some embodiments, the amino acid recognition molecule is soluble in the composition.

[0020] In some embodiments, the present application provides a compound of formula (II): AY 1 -D (II) where A is an amino acid binding moiety comprising at least one amino acid recognition molecule; Y 1 is a nucleic acid or a polypeptide; and D is a labeling moiety comprising at least one detectable label. In some embodiments, the amino acid recognition molecule is 1 When Y is a nucleic acid, the nucleic acid forms a covalent or non-covalent linking group. 1 is a polypeptide, the polypeptide is 50×10 -9 Dissociation constant (K D In some embodiments, K D is 1 x 10 -9Less than M, 1×10 -10 Less than M, 1×10 -11 Less than M or 1×10 -12 Less than M.

[0021] In some aspects, the present application provides an amino acid recognition molecule comprising a nucleic acid, at least one amino acid recognition molecule attached to a first attachment site on the nucleic acid, and at least one detectable label attached to a second attachment site on the nucleic acid, wherein the nucleic acid forms a covalent or non-covalent linking group between the at least one amino acid recognition molecule and the at least one detectable label. In some embodiments, the nucleic acid comprises a first oligonucleotide strand. In some embodiments, the nucleic acid further comprises a second oligonucleotide strand hybridized to the first oligonucleotide strand.

[0022] In some aspects, the present application provides an amino acid recognition molecule comprising a multivalent protein comprising at least two ligand binding sites, at least one amino acid recognition molecule attached to the protein via a first ligand moiety bound to a first ligand binding site on the protein, and at least one detectable label attached to the protein via a second ligand moiety bound to a second ligand binding site on the protein. In some embodiments, the multivalent protein is an avidin protein.

[0023] In some embodiments, the shielding amino acid recognition molecule may be used in a polypeptide sequencing method according to the present application or any method known in the art. Thus, in some aspects, the present application provides a polypeptide sequencing method comprising contacting a polypeptide molecule with one or more shielding amino acid recognition molecules according to the present application (e.g., in an Edman-type degradation reaction, in a kinetic sequencing reaction, or other method known in the art). For example, in some embodiments, the method comprises contacting a polypeptide molecule with at least one amino acid recognition molecule comprising a shield or shielding element according to the present application, and detecting association of the at least one amino acid recognition molecule with the polypeptide molecule.

[0024] In some aspects, the present application provides a method for identifying a protein of interest in a mixed sample. In some embodiments, the method includes cleaving the mixed protein sample to generate a plurality of polypeptide fragments. In some embodiments, the method further includes determining the amino acid sequence of at least one of the plurality of polypeptide fragments by a method according to the present application. In some embodiments, the method further includes identifying the protein of interest in the mixed sample if the amino acid sequence is uniquely identifiable to the protein of interest.

[0025] In some embodiments, the method of identifying a protein of interest in a mixed sample includes cleaving the mixed protein sample to generate a plurality of polypeptide fragments. In some embodiments, the method further includes labeling one or more types of amino acids in the plurality of polypeptide fragments with one or more different luminescent labels. In some embodiments, the method further includes measuring luminescence over time for at least one of the plurality of labeled polypeptides. In some embodiments, the method further includes determining the amino acid sequence of the at least one labeled polypeptide based on the detected luminescence. In some embodiments, the method further includes identifying the protein of interest in the mixed sample if the amino acid sequence is uniquely identifiable to the protein of interest.

[0026] Thus, in some embodiments, the subject polypeptide molecules or proteins analyzed herein may be a mixed or purified sample. In some embodiments, the subject polypeptide molecules or proteins are obtained from a biological sample (e.g., blood, tissue, saliva, urine, or other biological source). In some embodiments, the subject polypeptide molecules or proteins are obtained from a patient sample (e.g., a human sample).

[0027] Details of certain embodiments of the invention are set forth in the Detailed Description of Certain Embodiments set forth below. Other features, objects, and advantages of the invention will become apparent from the definition, examples, drawings, and claims.

[0028] Those skilled in the art will appreciate that the drawings described herein are for illustrative purposes only. It should be understood that in some instances, various aspects of the invention may be shown exaggerated or enlarged to facilitate understanding of the invention. In the drawings, like reference characters generally refer to like features, functionally similar elements, and / or structurally similar elements throughout the various drawings. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating the principles of the teachings. The drawings are not intended to limit the scope of the present teachings in any way.

[0029] The features and advantages of the present invention will become more apparent from the detailed description set forth below when considered in conjunction with the drawings. When describing the embodiments with reference to the drawings, directional references (such as "above," "below," "top," "bottom," "left," "right," "horizontal," "vertical," etc.) may be used. Such references are merely intended to assist the reader in viewing the drawings in a normal orientation. These directional references are not intended to describe a preferred or only orientation of an embodied device. The device may be embodied in other orientations.

[0030] As will be apparent from the detailed description, the examples depicted in the figures and further described for purposes of illustration throughout this application illustrate non-limiting embodiments, and in some cases may simplify certain processes or omit features or steps for purposes of clearer illustration. [Brief description of the drawings]

[0031] [Figure 1A] An example of polypeptide sequencing with detection (FIG. 1A) and analysis (FIG. 1B) of single molecule binding interactions is shown. [Figure 1B] An example of polypeptide sequencing with detection (FIG. 1A) and analysis (FIG. 1B) of single molecule binding interactions is shown. [Figure 1C] Various examples of labeled affinity reagents and methods of use according to the present application are shown: Figure 1C depicts exemplary configurations of labeled affinity reagents, including labeled enzymes and labeled aptamers, that selectively bind to one or more types of terminal amino acids. [Figure 1D] Various examples of labeled affinity reagents and methods of use according to the present application are shown: Figure ID generally depicts a degradation-based process for polypeptide sequencing using labeled affinity reagents. [Figure 1E] Various examples of labeled affinity reagents and methods of use according to the present application are shown: Figure 1E shows an example of polypeptide sequencing using labeled aptamers by repeated cycles of terminal amino acid detection, modification, and cleavage. [Diagram 2] An example of real-time polypeptide sequencing is shown using labeled exopeptidases that each selectively bind to and cleave different types of terminal amino acids. [Figure 3A] Figure 3A shows an example of real-time polypeptide sequencing by evaluating the binding interactions of terminal and / or internal amino acids with a labeled affinity reagent and a labeled cleavage reagent (e.g., a labeled non-specific exopeptidase). Figure 3B shows an example of real-time sequencing by detecting a series of pulses at the signal output. [Figure 3B]Figure 3B shows an example of real-time polypeptide sequencing by evaluating the binding interactions of terminal and / or internal amino acids with a labeled affinity reagent and a labeled cleavage reagent (e.g., a labeled non-specific exopeptidase). Figure 3B depicts a schematic of the temperature-dependent sequencing process. [Figure 4] An example of real-time polypeptide sequencing by assessing the binding interactions of terminal and internal amino acids with a labeled affinity reagent and a labeled non-specific exopeptidase is shown. [Figure 5A] Non-limiting examples of affinity reagents labeled via shielding elements are shown in Figure 5A, which illustrates single molecule peptide sequencing with affinity reagents labeled via conventional covalent linkages. [Figure 5B] Figure 5B illustrates a non-limiting example of an affinity reagent labeled via a shielding element. Figure 5B illustrates single molecule peptide sequencing with an affinity reagent that includes a shielding element. [Figure 5C] 5C-5E illustrate various examples of shielding elements according to the present application. [Figure 5D] 5C-5E illustrate various examples of shielding elements according to the present application. [Figure 5E] 5C-5E illustrate various examples of shielding elements according to the present application. [Figure 6] 1 shows an example of identifying a polypeptide based on a unique combination of amino acids detected in a labeled polypeptide. [Figure 7] 1 shows an example of polypeptide sequencing by detecting the emission of light from a labeled polypeptide that is subjected to repeated cycles of terminal amino acid modification and cleavage. [Figure 8A]Figure 8A shows an example of polypeptide sequencing of a labeled polypeptide by processive enzymatic cleavage. Figure 8B shows an example of sequencing by processive enzymatic cleavage of a labeled polypeptide by a fixed terminal peptidase. [Figure 8B] Figure 8B shows an example of polypeptide sequencing of a labeled polypeptide by processive enzymatic cleavage. Figure 8B shows an example of sequencing by processive enzymatic cleavage of an immobilized labeled polypeptide by terminal peptidase. [Figure 8C] Figure 8C shows an example of polypeptide sequencing of a labeled polypeptide by processive enzymatic cleavage. Figure 8C illustrates a schematic example of a real-time sequencing process carried out according to Figure 8B. [Figure 9] Schematic illustrating an example of sequencing by cofactor-based FRET using an immobilized ATP-dependent protease of a polypeptide substrate, donor-labeled ATP, and acceptor-labeled amino acids. [Figure 10A]

[0033] Figure 10 shows various examples of sample and sample well surface preparations for the analysis of polypeptides and proteins according to the present application: Figure 10A generally depicts an example process for preparing terminally modified polypeptides from a protein sample. [Figure 10B] 10A-10C show various exemplary preparations of samples and sample well surfaces for analysis of polypeptides and proteins according to the present application.FIG 10B generally depicts an exemplary process for conjugating a solubilizing linker to a polypeptide. [Figure 10C] Various examples of sample and sample well surface preparations for the analysis of polypeptides and proteins according to the present application are shown: Figure 10C shows an example schematic of a sample well with a modified surface that can be used to facilitate single molecule immobilization on the bottom surface. [Figure 11] FIG. 1 is a diagram of an exemplary sequence data processing pipeline for analyzing data obtained during a polypeptide degradation process according to some embodiments of the technology described herein. [Figure 12] 1 is a flow chart of an exemplary process for determining the amino acid sequence of a polypeptide molecule according to some embodiments of the technology described herein. [Figure 13] 1 is a flow chart of an exemplary process for determining an amino acid sequence corresponding to a polypeptide according to some embodiments of the technology described herein. [Figure 14] FIG. 1 is a block diagram of an example computer system that may be used to implement some embodiments of the techniques described herein. [Figure 15A] Figure 15 shows experimental data for selected peptide-linker conjugates that were prepared and evaluated for the solubility enhancement offered by various solubilizing linkers. Figure 15A shows example structures of peptide-linker conjugates that were synthesized and evaluated. [Figure 15B] Figure 15B shows experimental data for selected peptide-linker conjugates that were prepared and evaluated for the solubility enhancement offered by various solubilizing linkers. Figure 15B shows LCMS results demonstrating N-terminal peptide cleavage. [Figure 15C] Figure 15C shows experimental data for selected peptide-linker conjugates that were prepared and evaluated for the solubility enhancement offered by various solubilizing linkers. Figure 15C shows the results of a loading experiment. [Figure 16] 1 shows a summary of the amino acid cleavage activities of selected exopeptidases based on experimental results. [Figure 17A] 17A shows experimental data of a dye / peptide conjugate assay for detection and cleavage of terminal amino acids. FIG. 17A shows an example scheme and example structures used to perform the dye / peptide conjugate assay. [Figure 17B] Figure 17B shows experimental data of a dye / peptide conjugate assay for detection and cleavage of terminal amino acids.Figure 17B shows imaging results of peptide-linker conjugate loading into sample wells in an on-chip assay. [Figure 17C] 17A and 17B show experimental data of a dye / peptide conjugate assay for detection and cleavage of terminal amino acids. FIG. 17C shows an example signal trace detecting peptide conjugate loading and terminal amino acid cleavage. [Figure 18A] 18A shows experimental data of a FRET dye / peptide conjugate assay for detection and cleavage of terminal amino acids. FIG. 18A shows an example scheme and example structures used to perform a FRET dye / peptide conjugate assay. [Figure 18B] 18A shows experimental data of a FRET dye / peptide conjugate assay for detection and cleavage of terminal amino acids. FIG. 18B shows FRET imaging results at various time points. [Figure 18C] 18A and 18B show experimental data of a FRET dye / peptide conjugate assay for detection and cleavage of terminal amino acids. FIG. 18C shows the cutting efficiency at various time points. [Figure 18D] 18A-18C show experimental data for a FRET dye / peptide conjugate assay for detection and cleavage of terminal amino acids. FIG. 18D shows the cuts indicated at each of the various time points. [Figure 18E] Experimental data from a FRET dye / peptide conjugate assay for detection and cleavage of terminal amino acids are shown. Figure 18E shows additional FRET imaging results at various time points with proline iminopeptidase (yPIP) from Yersinia pestis. [Figure 18F] Experimental data of FRET dye / peptide conjugate assay for detection and cleavage of terminal amino acids are shown. Figure 18F shows FRET imaging results at various time points with aminopeptidase (VPr) from Vibrio proteolyticus. [Figure 19A] 19A shows experimental data of terminal amino acid discrimination by labeled affinity reagents, and FIG. 19A shows the crystal structure of the ClpS2 protein labeled for these experiments. [Figure 19B] 19A and 19B show experimental data of terminal amino acid discrimination by labeled affinity reagents, and FIGURE 19B shows single molecule intensity traces illustrating N-terminal amino acid discrimination by labeled ClpS2 protein. [Figure 19C]19A-C show experimental data for terminal amino acid discrimination by labeled affinity reagents, and FIGURE 19C is a plot showing the average pulse duration for various terminal amino acids. [Figure 19D] 19A and 19B show experimental data for terminal amino acid discrimination by labeled affinity reagents, and FIG. 19D is a plot showing the average inter-pulse duration for various terminal amino acids. [Figure 19E] 19A and 19B show experimental data of terminal amino acid discrimination by labeled affinity reagents, and FIG. 19E shows plots further illustrating discriminant pulse durations between various terminal amino acids. [Figure 19F] Experimental data on terminal amino acid recognition by labeled affinity reagents are shown in Figures 19F, 19G, and 19H, which show example results from retention time analysis demonstrating leucine recognition by the ClpS protein (teClpS) from Thermosynochoccus elongatus. [Figure 19G] Experimental data on terminal amino acid recognition by labeled affinity reagents are shown in Figures 19F, 19G, and 19H, which show example results from retention time analysis demonstrating leucine recognition by the ClpS protein (teClpS) from Thermosynochoccus elongatus. [Figure 19H] Experimental data on terminal amino acid recognition by labeled affinity reagents are shown in Figures 19F, 19G, and 19H, which show example results from retention time analysis demonstrating leucine recognition by the ClpS protein (teClpS) from Thermosynochoccus elongatus. [Figure 19I] 19 shows experimental data of terminal amino acid discrimination by labeled affinity reagents. FIG. 19I shows example results of retention time analysis demonstrating the distinguishable recognition of phenylalanine, leucine, tryptophan, and tyrosine by A. tumefaciens ClpS1. [Figure 19J]19A-19J show experimental data of terminal amino acid discrimination by labeled affinity reagents. FIG. 19J shows example results of retention time analysis demonstrating leucine recognition by S. elongatus ClpS2. [Figure 19K] 19K to 19L show experimental data on terminal amino acid recognition by a labeled affinity reagent, and show examples of retention time analysis results demonstrating proline recognition by GID4. [Figure 19L] 19K to 19L show experimental data on terminal amino acid recognition by a labeled affinity reagent, and show examples of retention time analysis results demonstrating proline recognition by GID4. [Figure 20A] Figure 20 shows example results of polypeptide sequencing reactions performed in real time with a labeled ClpS2 recognition protein and an aminopeptidase cleavage reagent in the same reaction mixture: Figure 20A shows signal trace data of the first sequencing reaction. [Figure 20B] Figure 20B shows example results of polypeptide sequencing reactions performed in real time with a labeled ClpS2 recognition protein and an aminopeptidase cleavage reagent in the same reaction mixture. Figure 20B shows pulse duration statistics of the signal trace data shown in Figure 20A. [Figure 20C] Figure 20C shows example results of a polypeptide sequencing reaction performed in real time with a labeled ClpS2 recognition protein and an aminopeptidase cleavage reagent in the same reaction mixture, Figure 20D shows signal trace data of the second sequencing reaction. [Figure 20D] Figure 20D shows example results of a polypeptide sequencing reaction performed in real time with a labeled ClpS2 recognition protein and an aminopeptidase cleavage reagent in the same reaction mixture. Figure 20D shows pulse duration statistics of the signal trace data shown in Figure 20C. [Figure 21A] Experimental data on terminal amino acid identification and cleavage by labeled exopeptidases are shown, and Figure 21A shows the crystal structure of site-specifically labeled proline iminopeptidase (yPIP) for these experiments. [Figure 21B]Figure 21B shows experimental data of terminal amino acid identification and cleavage by labeled exopeptidase, and Figure 21B shows the degree of labeling of purified protein products. [Figure 21C] Experimental data of terminal amino acid identification and cleavage by labeled exopeptidase are shown. Figure 21C is an image of an SDS page confirming site-specific labeling of yPIP. [Figure 21D] Experimental data of terminal amino acid identification and cleavage by labeled exopeptidase are shown. Figure 21D is an overexposed image of an SDS page gel confirming site-specific labeling. [Figure 21E] Experimental data of terminal amino acid identification and cleavage by labeled exopeptidase is shown. Figure 21E is an image of a Coomassie stained gel confirming the purity of the labeled protein product. [Figure 21F] The experimental data of terminal amino acid identification and cleavage by labeled exopeptidase are shown. Figure 21F is an HPLC trace demonstrating the cleavage activity of labeled exopeptidase. The sequence YPYPYPK corresponds to SEQ ID NO: 82. The sequence PYPYPK corresponds to SEQ ID NO: 83. [Figure 22A] 22A shows data from experiments assessing the recognition of amino acids containing specific post-translational modifications. FIG. 22A shows a representative trace demonstrating phosphotyrosine recognition by an SH2 domain-containing protein. [Figure 22B] Figure 22B shows data from an experiment assessing the recognition of amino acids containing specific post-translational modifications. Figure 22B shows pulse duration data corresponding to the traces in Figure 22A, and Figure 22C shows statistics determined for the traces. [Figure 22C] Figure 22B shows data from an experiment assessing the recognition of amino acids containing specific post-translational modifications. Figure 22B shows pulse duration data corresponding to the traces in Figure 22A, and Figure 22C shows statistics determined for the traces. [Figure 22D] 22D-22F show data from experiments assessing the recognition of amino acids containing specific post-translational modifications. Figures 22D-22F show representative traces from negative control experiments. [Figure 22E]22D-22F show data from experiments assessing the recognition of amino acids containing specific post-translational modifications. Figures 22D-22F show representative traces from negative control experiments. [Figure 22F] 22D-22F show data from experiments assessing the recognition of amino acids containing specific post-translational modifications. Figures 22D-22F show representative traces from negative control experiments. [Figure 23] 1 is a plot showing the median pulse duration from experiments evaluating the effect of penultimate amino acids on pulse duration. [Figure 24A] 24A shows data from an experiment assessing simultaneous amino acid recognition by differentially labeled recognition molecules. Figure 24A shows a representative trace. [Figure 24B] Figure 24B shows data from experiments assessing simultaneous amino acid recognition by differentially labeled recognition molecules. Figure 24B is a plot comparing the pulse duration data obtained during these experiments for each recognition molecule. [Figure 24C] Figure 24C shows data from experiments assessing simultaneous amino acid recognition by differentially labeled recognition molecules, and Figure 24D shows pulse duration statistics for these experiments. [Figure 25A] 25A shows data from an experiment assessing the photostability of peptides upon single molecule recognition. Figure 25A shows a representative trace of recognition with atClpS2-V1 labeled with a dye approximately 2 nm from the amino acid binding site. [Figure 25B] Figure 25B shows data from experiments assessing the photostability of peptides upon single molecule recognition, and Figure 25B shows a visualization of the structure of the ClpS2 protein used in these experiments. [Figure 25C] Data from experiments assessing the photostability of peptides upon single molecule recognition are shown in Figure 25C, which shows a representative trace of recognition with ClpS2 labeled with a dye >10 nm from the amino acid binding site via a DNA / protein linker. [Figure 26A] Representative traces of polypeptide sequencing reactions performed in real time on a complementary metal-oxide-semiconductor (CMOS) chip using ClpS2 recognition protein labeled via a DNA / streptavidin linker in the presence of an aminopeptidase cleavage reagent are shown. [Figure 26B] Representative traces of polypeptide sequencing reactions performed in real time on a complementary metal-oxide-semiconductor (CMOS) chip using ClpS2 recognition protein labeled via a DNA / streptavidin linker in the presence of an aminopeptidase cleavage reagent are shown. [Figure 26C] Representative traces of polypeptide sequencing reactions performed in real time on a complementary metal-oxide-semiconductor (CMOS) chip using ClpS2 recognition protein labeled via a DNA / streptavidin linker in the presence of an aminopeptidase cleavage reagent are shown. [Figure 26D] Representative traces of polypeptide sequencing reactions performed in real time on a complementary metal-oxide-semiconductor (CMOS) chip using ClpS2 recognition protein labeled via a DNA / streptavidin linker in the presence of an aminopeptidase cleavage reagent are shown. [Figure 27] Representative traces of polypeptide sequencing reactions performed in real time with atClpS2-V1 recognition protein labeled via a DNA / streptavidin linker in the presence of Pyrococcus horikoshii TET aminopeptidase cleavage reagent are shown. [Figure 28A] Representative trace data of polypeptide sequencing reactions performed in real time using multiple types of exopeptidases with differential cleavage specificities are shown. Figure 28A shows a representative trace of a reaction performed with hTET exopeptidase with the expanded pulse pattern region shown in Figure 28B. The sequence YAAWAAFADDDWK in Figure 28A corresponds to SEQ ID NO:78. [Figure 28B] Representative trace data of polypeptide sequencing reactions performed in real time using multiple types of exopeptidases with differential cleavage specificities are shown. Figure 28A shows a representative trace of a reaction performed with hTET exopeptidase with the expanded pulse pattern region shown in Figure 28B. The sequence YAAWAAFADDDWK in Figure 28A corresponds to SEQ ID NO:78. [Figure 28C] Representative trace data of polypeptide sequencing reactions performed in real time using multiple types of exopeptidases with differential cleavage specificities are shown. Figure 28C shows representative traces of reactions performed with both hTET and yPIP exopeptidases, with the expanded pulse pattern region shown in Figure 28D and additional representative traces shown in Figure 28E. The sequence FYPLPWPDDDYK in Figure 28C corresponds to SEQ ID NO:80. [Figure 28D] Representative trace data of polypeptide sequencing reactions performed in real time using multiple types of exopeptidases with differential cleavage specificities are shown. Figure 28C shows representative traces of reactions performed with both hTET and yPIP exopeptidases, with the expanded pulse pattern region shown in Figure 28D and additional representative traces shown in Figure 28E. The sequence FYPLPWPDDDYK in Figure 28C corresponds to SEQ ID NO:80. [Figure 28E] Representative trace data of polypeptide sequencing reactions performed in real time using multiple types of exopeptidases with differential cleavage specificities are shown. Figure 28C shows representative traces of reactions performed with both hTET and yPIP exopeptidases, with the expanded pulse pattern region shown in Figure 28D and additional representative traces shown in Figure 28E. The sequence FYPLPWPDDDYK in Figure 28C corresponds to SEQ ID NO:80. [Figure 28F] Figure 28F shows representative traces of further reactions performed with both hTET and yPIP exopeptidases, with the expanded pulse pattern region shown in Figure 28G and additional representative traces shown in Figure 28H. The sequence YPLPWPDDDYK in Figures 28F and 28I corresponds to SEQ ID NO:81. [Figure 28G] Figure 28F shows representative traces of further reactions performed with both hTET and yPIP exopeptidases, with the expanded pulse pattern region shown in Figure 28G and additional representative traces shown in Figure 28H. The sequence YPLPWPDDDYK in Figures 28F and 28I corresponds to SEQ ID NO:81. [Fig. 28H] Figure 28F shows representative traces of further reactions performed with both hTET and yPIP exopeptidases, with the expanded pulse pattern region shown in Figure 28G and additional representative traces shown in Figure 28H. The sequence YPLPWPDDDYK in Figures 28F and 28I corresponds to SEQ ID NO:81. [Figure 28I] Representative trace data of polypeptide sequencing reactions performed in real time using multiple types of exopeptidases with differential cleavage specificities are shown. Figure 28I shows representative traces of reactions performed with both PfuTET and yPIP exopeptidases with the expanded pulse pattern region shown in Figure 28J. The sequence YPLPWPDDDYK in Figures 28F and 28I corresponds to SEQ ID NO:81. [Figure 28J] Representative trace data of polypeptide sequencing reactions performed in real time using multiple types of exopeptidases with differential cleavage specificities are shown. Figure 28I shows representative traces of reactions performed with both PfuTET and yPIP exopeptidases with the expanded pulse pattern region shown in Figure 28J. The sequence YPLPWPDDDYK in Figures 28F and 28I corresponds to SEQ ID NO:81. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0032] Aspects of the present application relate to methods of protein sequencing and identification, methods of polypeptide sequencing and identification, methods of amino acid identification, and compositions for carrying out such methods.

[0033] In some aspects, the present application relates to the discovery of polypeptide sequencing techniques that can be implemented using existing analytical equipment with little or no device modification. For example, previous polypeptide sequencing strategies have involved repeated cycling of various reagent mixtures through a reaction vessel containing the polypeptide to be analyzed. Such strategies may require modification of existing analytical equipment, such as nucleic acid sequencing equipment, which may not be equipped with a flow cell or similar device capable of reagent cycling. The inventors have recognized and appreciated that certain polypeptide sequencing techniques of the present application do not require repeated reagent cycling, thereby allowing the use of existing equipment without significant modifications that may increase the size of the equipment. Thus, in some aspects, the present application provides polypeptide sequencing methods that allow the use of smaller sequencing equipment. In some aspects, the present application relates to the discovery of polypeptide sequencing techniques that allow both genomic and proteomic analysis to be performed using the same sequencing equipment.

[0034] The inventors further recognized and appreciated that differential binding interactions can provide an additional or alternative approach to conventional labeling strategies for polypeptide sequencing. Conventional polypeptide sequencing can involve labeling each type of amino acid with a unique identifiable label. With at least 20 different types of naturally occurring amino acids, as well as numerous post-translational variations, such a process can be laborious and error-prone. In some aspects, the present application relates to the discovery of techniques that involve the use of amino acid recognition molecules that differentially associate with different types of amino acids to generate detectable characteristic signatures that are indicative of the amino acid sequence of a polypeptide. Thus, aspects of the present application provide techniques that do not require polypeptide labeling and / or harsh chemical reagents used in certain conventional polypeptide sequencing, thereby increasing the throughput and / or accuracy of sequence information obtained from a sample.

[0035] In some aspects, the present application relates to the discovery that a polypeptide sequencing reaction can be monitored in real time using only a single reaction mixture (e.g., without the need for repeated reagent cycling through a reaction vessel). As detailed above, a conventional polypeptide sequencing reaction can include exposing a polypeptide to various reagent mixtures to cycle between an amino acid detection step and an amino acid cleavage step. Thus, in some aspects, the present application relates to an advancement in next-generation sequencing that allows polypeptide analysis by real-time amino acid detection throughout an ongoing degradation reaction. Such an approach to polypeptide analysis by dynamic sequencing is described below.

[0036] As described herein, in some aspects, the present application provides methods for sequencing a polypeptide by obtaining data during a polypeptide degradation process and analyzing the data to determine a portion of the data corresponding to amino acids that are sequentially exposed at a terminus of the polypeptide during the degradation process. In some embodiments, the portion of the data comprises a series of signal pulses indicative of the association of one or more amino acid recognition molecules with sequential amino acids that are sequentially exposed at a terminus of the polypeptide (e.g., during degradation). In some embodiments, the series of signal pulses corresponds to a series of reversible single molecule binding interactions at a terminus of the polypeptide during the degradation process.

[0037] A non-limiting example of polypeptide sequencing by detecting single molecule binding interactions during the polypeptide degradation process is illustrated diagrammatically in FIG. 1A. An example signal trace (I) is shown in a series of panels (II) depicting various association events as they correspond to changes in the signal. As shown, association events between amino acid recognition molecules (in the form of spots) and terminal amino acids of a polypeptide (shown as beads-on-a-string) generate changes in signal magnitude that persist for a certain duration.

[0038] Panels (A) and (B) depict various association events between an amino acid recognition molecule and a first amino acid (e.g., the first terminal amino acid) exposed at the terminus of a polypeptide. Each association event generates a change in signal trace (I) characterized by a change in signal magnitude that persists for the duration of the association event. Thus, the duration between association events in panels (A) and (B) corresponds to a duration during which the polypeptide is not detectably associated with the amino acid recognition molecule.

[0039] Panels (C) and (D) depict various association events between an amino acid recognition molecule and a second amino acid (e.g., a second terminal amino acid) exposed at the terminus of a polypeptide. As described herein, an amino acid that is "exposed" at the terminus of a polypeptide is an amino acid that is still attached to the polypeptide and that, upon degradation, becomes a terminal amino acid (e.g., either alone or with one or more additional amino acids) upon removal of the preceding terminal amino acid. Thus, the first and second amino acids in the series of panel (II) provide illustrative examples of sequential amino acids exposed at the terminus of a polypeptide, where the second amino acid is the one that became the terminal amino acid upon removal of the first amino acid.

[0040] As generally depicted, the association events in panels (C) and (D) generate changes in signal trace (I) characterized by a change in magnitude that persists for a relatively shorter duration than those in panels (A) and (B), and the duration between the association events in panels (C) and (D) is relatively shorter than that of panels (A) and (B). As described herein, in some embodiments, either one or both of these distinguishable changes in signal can be used to determine a characteristic pattern in signal trace (I) that is capable of distinguishing between different types of amino acids. In some embodiments, a transition from one characteristic pattern to another characteristic pattern is indicative of an amino acid cleavage. As used herein, in some embodiments, an amino acid cleavage refers to the removal of at least one amino acid from the terminus of a polypeptide (e.g., removal of at least one terminal amino acid from a polypeptide). In some embodiments, an amino acid cleavage is determined by inference based on the duration between the characteristic patterns. In some embodiments, an amino acid cleavage is determined by detecting a change in signal generated by the association of a labeled cleavage reagent with an amino acid at the terminus of the polypeptide. As amino acids are sequentially cleaved from the ends of the polypeptide during degradation, a series of changes in size, or a series of signal pulses, is detected, in some embodiments, the signal pulse data can be analyzed as illustrated in FIG.

[0041] In some embodiments, the signal data can be analyzed to extract signal pulse information by applying a threshold level to one or more parameters of the signal data. For example, panel (III) shows a threshold magnitude level ("M") applied to the signal data of example signal trace (I). L In some embodiments, M L is the minimum difference between the signal detected at a time point and the baseline determined for a given data set. In some embodiments, a signal pulse ("sp") is LThe threshold duration is assigned to each portion of data that is indicative of a change in magnitude exceeding M L to determine whether a signal pulse is assigned to that portion. For example, an experimental artifact may not persist for a sufficient duration to attribute a signal pulse with the desired confidence. L (e.g., transient association events that may not distinguish amino acid types, non-specific detection events such as reagents diffusing into or sticking within the observation region). Therefore, in some embodiments, signal pulses are extracted from the signal data based on a threshold magnitude level and a threshold duration.

[0042] The extracted signal pulse information is shown in panel (III) with an example signal trace (I) overlaid for illustrative purposes. In some embodiments, the signal pulse magnitude peak is M L It should be appreciated that in some embodiments, a "signal pulse" as used herein can refer to a change in signal data (e.g., raw signal data exemplified by example signal trace (I)) or signal pulse information extracted therefrom (e.g., processed signal data exemplified in panel (IV)) that persists above the baseline for a duration.

[0043] Panel (IV) shows signal pulse information extracted from the example signal trace (I). In some embodiments, the signal pulse information can be analyzed to identify different types of amino acids based on various characteristic patterns in a series of signal pulses. For example, as shown in panel (IV), the signal pulse information identifies different types of amino acids based on a first characteristic pattern ("CP 1 ") based on a first type of amino acid and a second characteristic pattern ("CP 2") are indicative of a second type of amino acid. For example, two signal pulses detected at earlier time points are indicative of CP 1 The two signal pulses detected at a later time point provide information indicative of the first amino acid at the end of the polypeptide based on the CP 2 This provides information indicative of the second amino acid at the end of the polypeptide based on the above.

[0044] Also shown in panel (IV), each signal pulse includes a pulse duration ("pd") corresponding to an association event between the amino acid recognition molecule and an amino acid of the characteristic pattern. In some embodiments, the pulse duration is characteristic of the dissociation rate of the binding. Also shown, each signal pulse of the characteristic pattern is separated from other signal pulses of the characteristic pattern by an interpulse duration ("ipd"). In some embodiments, the interpulse duration is characteristic of the association rate of the binding. In some embodiments, a change in magnitude ("ΔM") can be determined for the signal pulse based on the difference between the baseline and the peak of the signal pulse. In some embodiments, the characteristic pattern is determined based on the pulse duration. In some embodiments, the characteristic pattern is determined based on the pulse duration and the interpulse duration. In some embodiments, the characteristic pattern is determined based on any one or more of the pulse duration, the interpulse duration, and the change in magnitude.

[0045] Thus, as illustrated by Figures 1A-1B, in some embodiments, polypeptide sequencing is performed by detecting a series of signal pulses indicative of the association of one or more amino acid recognition molecules with successive amino acids exposed at the termini of a polypeptide in an ongoing cleavage reaction. The series of signal pulses can be analyzed to determine a characteristic pattern in the series of signal pulses, and the change in the characteristic pattern over time can be used to determine the amino acid sequence of the polypeptide.

[0046] In some embodiments, the series of signal pulses comprises a series of changes in the magnitude of the optical signal over time. In some embodiments, the series of changes in the optical signal comprises a series of changes in the luminescence generated upon the association event. In some embodiments, the luminescence is generated by a detectable label associated with one or more reagents of the sequencing reaction. For example, in some embodiments, each of the one or more amino acid recognition molecules comprises a luminescent label. In some embodiments, the cleavage reagent comprises a luminescent label. Examples of luminescent labels and their uses according to the present application are provided elsewhere herein.

[0047] In some embodiments, the series of signal pulses comprises a series of changes in the magnitude of the electrical signal over time. In some embodiments, the series of changes in the electrical signal comprises a series of changes in the conductance generated during the association event. In some embodiments, the conductivity is generated by a detectable label associated with one or more reagents of the sequencing reaction. For example, in some embodiments, each of the one or more amino acid recognition molecules comprises a conductivity label. Examples of conductivity labels and their uses according to the present application are provided elsewhere herein. Methods for identifying single molecules using conductivity labels have been described (see, for example, US Patent Application Publication No. 2017 / 0037462).

[0048] In some embodiments, the series of changes in conductance includes a series of changes in conductance through a nanopore. For example, methods for evaluating receptor-ligand interactions using nanopores have been described (see, for example, Thakur, AK and Movileanu, L., 2019, Nature Biotechnology, vol. 37, no. 1). The inventors have recognized and appreciated that such nanopores may be used to monitor polypeptide sequencing reactions according to the present application. Thus, in some embodiments, the present application provides a method of polypeptide sequencing comprising contacting a single polypeptide molecule with one or more amino acid recognition molecules, where the single polypeptide molecule is immobilized in a nanopore. In some embodiments, the method further comprises sequencing the single polypeptide molecule by detecting a series of changes in conductance through the nanopore indicative of association of one or more terminal amino acid recognition molecules with sequential amino acids exposed at the termini of the single polypeptide as the single polypeptide is degraded.

[0049] In some aspects, the present application provides methods for sequencing and / or identifying individual proteins in a complex mixture of proteins by identifying one or more types of amino acids of a polypeptide from the mixture. In some embodiments, one or more amino acids of a polypeptide (e.g., terminal amino acids and / or internal amino acids) are labeled (e.g., directly or indirectly, e.g., using a binding agent such as an amino acid recognition molecule) and the relative position of the labeled amino acid in the polypeptide is determined. In some embodiments, the relative position of the amino acid in the polypeptide is determined using a series of amino acid labeling and cleavage steps. However, in some embodiments, the relative position of the labeled amino acid in the polypeptide can be determined by translocating the labeled polypeptide through a pore (e.g., a protein channel) without removing the amino acid from the polypeptide and detecting a signal (e.g., a FRET signal) from the labeled amino acid upon translocation through the pore to determine the relative position of the labeled amino acid in the polypeptide molecule.

[0050] In some embodiments, the identity of a terminal amino acid (e.g., the N-terminal or C-terminal amino acid) is evaluated, then the terminal amino acid is removed, and the identity of the next terminal amino acid is evaluated, and this process is repeated until multiple successive amino acids in the polypeptide have been evaluated. In some embodiments, evaluating the identity of an amino acid includes determining the type of amino acid present. In some embodiments, determining the type of amino acid includes determining the identity of the actual amino acid, for example, by determining which of the 20 naturally occurring amino acids it is (e.g., using a binding agent specific for the individual terminal amino acid). In some embodiments, the type of amino acid is selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, selenocysteine, threonine, tryptophan, tyrosine, and valine.

[0051] However, in some embodiments, assessing the identity of the terminal amino acid type may include determining a subset of potential amino acids that may be present at the termini of the polypeptide. In some embodiments, this may be accomplished by determining that the amino acid is not one or more specific amino acids (and therefore may be any of the other amino acids). In some embodiments, this may be accomplished by determining (e.g., based on size, charge, hydrophobicity, post-translational modification, binding affinity) which of a particular subset of amino acids may be at the termini of the polypeptide (e.g., using a binding agent that binds to a particular subset of two or more terminal amino acids).

[0052] In some embodiments, assessing the identity of the terminal amino acid type includes determining that the amino acid comprises a post-translational modification, non-limiting examples of which include acetylation, ADP-ribosylation, caspase cleavage, citrullination, formylation, N-linked glycosylation, O-linked glycosylation, hydroxylation, methylation, myristoylation, neddylation, nitration, oxidation, palmitoylation, phosphorylation, prenylation, S-nitrosylation, sulfation, sumoylation, and ubiquitination.

[0053] In some embodiments, evaluating the identity of the terminal amino acid type includes determining that the amino acid comprises a side chain characterized by one or more biochemical properties. For example, the amino acid may comprise a non-polar aliphatic side chain, a positively charged side chain, a negatively charged side chain, a non-polar aromatic side chain, or a polar uncharged side chain. Non-limiting examples of amino acids comprising a non-polar aliphatic side chain include alanine, glycine, valine, leucine, methionine, and isoleucine. Non-limiting examples of amino acids comprising a positively charged side chain include lysine, arginine, and histidine. Non-limiting examples of amino acids comprising a negatively charged side chain include aspartic acid and glutamic acid. Non-limiting examples of amino acids comprising a non-polar aromatic side chain include phenylalanine, tyrosine, and tryptophan. Non-limiting examples of amino acids comprising a polar uncharged side chain include serine, threonine, cysteine, proline, asparagine, and glutamine.

[0054] In some embodiments, a protein or polypeptide can be digested into multiple smaller polypeptides, and sequence information can be obtained from one or more of these smaller polypeptides (e.g., using a method that involves sequentially evaluating the terminal amino acids of the polypeptide and removing that amino acid to expose the next amino acid at the terminus).

[0055] In some embodiments, a polypeptide is sequenced from its amino (N) terminus. In some embodiments, a polypeptide is sequenced from its carboxy (C) terminus. In some embodiments, a first terminus (e.g., the N- or C-terminus) of a polypeptide is fixed and the other terminus (e.g., the C- or N-terminus) is sequenced as described herein.

[0056] As used herein, sequencing a polypeptide means determining sequence information of a polypeptide. In some embodiments, this may involve determining the identity of each sequential amino acid of a portion (or all) of a polypeptide. However, in some embodiments, this may involve evaluating the identity of a subset of amino acids within a polypeptide (e.g., determining the relative positions of one or more amino acid types without determining the identity of each amino acid in the polypeptide). However, in some embodiments, it is possible to obtain amino acid content information from a polypeptide without directly determining the relative positions of various types of amino acids in the polypeptide. Amino acid content alone may be used to infer the identity of an existing polypeptide (e.g., by comparing the amino acid content to a database of polypeptide information and determining which polypeptides have the same amino acid content).

[0057] In some embodiments, sequence information of multiple polypeptide products obtained from a longer polypeptide or protein (e.g., via enzymatic and / or chemical cleavage) can be analyzed to reconstruct or infer the sequence of the longer polypeptide or protein.

[0058] Thus, in some embodiments, one or more types of amino acids are identified by detecting the emission of one or more labeled affinity reagents that selectively bind to one or more types of amino acids, hi some embodiments, one or more types of amino acids are identified by detecting the emission of a labeled polypeptide.

[0059] The inventors further recognized and appreciated that the polypeptide sequencing techniques described herein, particularly in contrast to conventional polypeptide sequencing techniques, may involve the generation of novel polypeptide sequencing data, such that conventional techniques for analyzing polypeptide sequencing data may be insufficient when applied to data generated using the polypeptide sequencing techniques described herein.

[0060] For example, conventional polypeptide sequencing techniques involving repeated reagent cycling may generate data associated with individual amino acids of the polypeptide being sequenced. In such cases, since the detected data corresponds to only one amino acid, analyzing the generated data would simply involve determining which amino acid is being detected at a particular time. In contrast, the polypeptide sequencing techniques described herein may generate data during a polypeptide degradation process while multiple amino acids of a polypeptide molecule are being detected, resulting in data in which it may be difficult to distinguish sections of data corresponding to different amino acids of a polypeptide. Therefore, the inventors have developed a new computational technique for analyzing such data generated by the polypeptide sequencing techniques described herein, including determining sections of data corresponding to individual amino acids, for example, by segmenting the data into portions corresponding to each amino acid association event. These sections can then be further analyzed to identify the amino acids detected during those individual sections.

[0061] As another example, conventional sequencing techniques involving the use of unique identifiable labels for each type of amino acid may simply involve analyzing which labels are detected at a particular time, without any consideration of the dynamics of how individual amino acids interact with other molecules. In contrast, the polypeptide sequencing techniques described herein generate data that are indicative of how amino acids interact with recognition molecules. As discussed above, the data may include a series of characteristic patterns that correspond to association events between amino acids and their respective recognition molecules. Therefore, the inventors have developed a new computational technique that analyzes the characteristic patterns to determine the type of amino acid that corresponds to that portion of data, allowing the determination of the amino acid sequence of a polypeptide by analyzing a series of different characteristic patterns.

[0062] Labeled affinity reagents and methods of use In some embodiments, the methods provided herein include contacting a polypeptide with an amino acid recognition molecule (also referred to herein as an amino acid recognition molecule that may or may not include a label) that selectively binds to one type of terminal amino acid. As used herein, in some embodiments, a terminal amino acid can refer to the amino terminal amino acid of a polypeptide or the carboxy terminal amino acid of a polypeptide. In some embodiments, the labeled affinity reagent selectively binds to one type of terminal amino acid over other types of terminal amino acids. In some embodiments, the labeled affinity reagent selectively binds to one type of terminal amino acid over internal amino acids of the same type. In still other embodiments, the labeled affinity reagent selectively binds to one type of amino acid anywhere in the polypeptide, e.g., the same type of amino acid as the terminal amino acid or the internal amino acid.

[0063] As used herein, in some embodiments, a type of amino acid refers to one of the 20 naturally occurring amino acids or a subset of the types. In some embodiments, a type of amino acid refers to a modified variant of one of the 20 naturally occurring amino acids or a subset of unmodified and / or modified variants thereof. Examples of modified amino acid variants include, but are not limited to, post-translational modification variants (e.g., acetylation, ADP-ribosylation, caspase cleavage, citrullination, formylation, N-linked glycosylation, O-linked glycosylation, hydroxylation, methylation, myristoylation, NEDDylation, nitration, oxidation, palmitoylation, phosphorylation, prenylation, S-nitrosylation, sulfation, SUMOylation, and ubiquitination), chemically modified variants, unnatural amino acids, and proteinogenic amino acids, such as selenocysteine ​​and pyrrolysine. In some embodiments, the subset of a type of amino acid includes more than 1 and less than 20 amino acids that have one or more similar biochemical properties. For example, in some embodiments, a type of amino acid refers to a type selected from amino acids having charged side chains (e.g., positively and / or negatively charged side chains), amino acids having polar side chains (e.g., polar uncharged side chains), amino acids having nonpolar side chains (e.g., nonpolar aliphatic and / or aromatic side chains), and amino acids having hydrophobic side chains.

[0064] In some embodiments, the methods provided herein include contacting a polypeptide with one or more labeled affinity reagents that selectively bind to one or more types of terminal amino acids. As an illustrative and non-limiting example, if four labeled affinity reagents are used in the methods of the present application, any one reagent selectively binds to one type of terminal amino acid that is different from the other one type of amino acid to which any of the other three selectively bind (e.g., a first reagent binds to a first type, a second reagent binds to a second type, a third reagent binds to a third type, and a fourth reagent binds to a fourth type of terminal amino acid). For purposes of this discussion, one or more labeled affinity reagents in the context of the methods described herein may alternatively be referred to as a set of labeled affinity reagents.

[0065] In some embodiments, the set of labeled affinity reagents includes at least one and up to six labeled affinity reagents. For example, in some embodiments, the set of labeled affinity reagents includes one, two, three, four, five, or six labeled affinity reagents. In some embodiments, the set of labeled affinity reagents includes ten or fewer labeled affinity reagents. In some embodiments, the set of labeled affinity reagents includes eight or fewer labeled affinity reagents. In some embodiments, the set of labeled affinity reagents includes six or fewer labeled affinity reagents. In some embodiments, the set of labeled affinity reagents includes four or fewer labeled affinity reagents. In some embodiments, the set of labeled affinity reagents includes three or fewer labeled affinity reagents. In some embodiments, the set of labeled affinity reagents includes two or fewer labeled affinity reagents. In some embodiments, the set of labeled affinity reagents includes four labeled affinity reagents. In some embodiments, the set of labeled affinity reagents includes at least two and up to 20 (e.g., at least two and up to 10, at least two and up to 8, at least four and up to 20, at least four and up to 10) labeled affinity reagents. In some embodiments, the set of labeled affinity reagents includes more than 20 (e.g., 20-25, 20-30) affinity reagents. However, it should be recognized that any number of affinity reagents may be used in accordance with the methods of the present application that is compatible with the desired use.

[0066] According to the present application, in some embodiments, one or more types of amino acids are identified by detecting the emission of a labeled affinity reagent (e.g., an amino acid recognition molecule that includes a luminescent label). In some embodiments, the labeled affinity reagent includes an affinity reagent that selectively binds to one type of amino acid and a luminescent label having an emission associated with the affinity reagent. Thus, the emission (e.g., emission lifetime, emission intensity, and other luminescent properties described elsewhere herein) can be associated with the selective binding of the affinity reagent to identify the amino acids of the polypeptide. In some embodiments, multiple types of labeled affinity reagents can be used in the present method, with each type including a luminescent label having an emission that is uniquely identifiable among the multiple. Suitable luminescent labels can include luminescent molecules such as fluorophore dyes and are described elsewhere herein.

[0067] In some embodiments, one or more types of amino acids are identified by detecting one or more electrical properties of the labeled affinity reagent. In some embodiments, the labeled affinity reagent includes an affinity reagent that selectively binds one type of amino acid and a conductivity label associated with the affinity reagent. Thus, one or more electrical properties (e.g., charge, current oscillation color, and other electrical properties) can be associated with the selective binding of the affinity reagent to identify the amino acids of the polypeptide. In some embodiments, multiple types of labeled affinity reagents can be used in the methods of the present application, with each type including a conductivity label that generates a uniquely identifiable change in electrical signal between the multiple types (e.g., conductance changes, e.g., characteristic patterns of conductivity magnitude changes and conductivity transitions). In some embodiments, the multiple types of labeled affinity reagents each include a conductivity label with a different number of charged groups (e.g., a different number of negatively and / or positively charged groups). Thus, in some embodiments, the conductivity label is a charge label. Examples of charge labels include dendrimers, nanoparticles, nucleic acids, and other polymers with multiple charged groups. In some embodiments, a conductivity label is uniquely identifiable by its net charge (eg, a net positive charge or a net negative charge), by its charge density, and / or by its charge group number.

[0068] In some embodiments, affinity reagents (e.g., amino acid recognition molecules) can be engineered by one of skill in the art using techniques known in the art. In some embodiments, the desired property can include the ability to selectively and with high affinity bind to one type of amino acid only when located at the termini of a polypeptide (e.g., N-terminus or C-terminus). In yet other embodiments, the desired property can include the ability to selectively and with high affinity bind to one type of amino acid when located at the termini of a polypeptide (e.g., N-terminus or C-terminus) and when located at internal positions of the polypeptide. In some embodiments, the desired property can include the ability to selectively and with low affinity bind to more than one type of amino acid (e.g., K of about 50 nM or more, e.g., about 50 nM to about 50 μM, about 100 nM to about 10 μM, about 500 nM to about 50 μM). D For example, in some embodiments, the present application provides methods of sequencing by detecting reversible binding interactions during the polypeptide degradation process. Advantageously, such methods can be performed using affinity reagents that reversibly bind with low affinity to more than one type of amino acid (e.g., a subset of amino acid types).

[0069] As used herein, in some embodiments, the terms "selective" and "specific" (and variations thereof, e.g., selectively, specifically, selectivity, specificity) refer to preferential binding interactions. For example, in some embodiments, a labeled affinity reagent that selectively binds one type of amino acid preferentially binds one type of amino acid over another type of amino acid. A selective binding interaction will typically discriminate between one type of amino acid (e.g., a terminal amino acid of one type) and another type of amino acid (e.g., a terminal amino acid of another type) by more than about 10-fold to 100-fold or more (e.g., more than about 1,000-fold or more than 10,000-fold). Thus, it should be appreciated that a selective binding interaction can refer to any binding interaction that uniquely identifies one type of amino acid over another type of amino acid. For example, in some aspects, the present application provides methods of polypeptide sequencing by obtaining data indicative of the association of one or more amino acid recognition molecules with a polypeptide molecule. In some embodiments, the data includes a series of signal pulses corresponding to a series of reversible amino acid recognition molecule binding interactions with amino acids of the polypeptide molecule, and the data can be used to determine the identity of the amino acids. Thus, in some embodiments, a "selective" or "specific" binding interaction refers to a detection binding interaction that discriminates between one type of amino acid and another type of amino acid.

[0070] In some embodiments, the labeled affinity reagent (e.g., amino acid recognition molecule) can be about 10 times more reactive than the labeled affinity reagent without significant binding to other types of amino acids. -6 Less than M (for example, about 10 -7 Less than M, about 10 -8 Less than M, about 10 -9 Less than M, about 10 -10 Less than M, about 10 -11 Less than M, about 10 -12 Less than M, 10 -16 Dissociation constant (K DIn some embodiments, the labeled affinity reagent selectively binds to one type of amino acid with a K of less than about 100 nM, less than about 50 nM, less than about 25 nM, less than about 10 nM, or less than about 1 nM. D In some embodiments, the labeled affinity reagent selectively binds to one type of amino acid (e.g., one type of terminal amino acid) at a K of about 50 nM to about 50 μM (e.g., about 50 nM to about 500 nM, about 50 nM to about 5 μM, about 500 nM to about 50 μM, about 5 μM to about 50 μM, or about 10 μM to about 50 μM). D In some embodiments, the labeled affinity reagent selectively binds one type of amino acid at a K of about 50 nM. D It selectively binds to one type of amino acid.

[0071] In some embodiments, the labeled affinity reagent (e.g., amino acid recognition molecule) is about 10 -6 Less than M (for example, about 10 -7 Less than M, about 10 -8 Less than M, about 10 -9 Less than M, about 10 -10 Less than M, about 10 -11 Less than M, about 10 -12 Less than M, 10 -16 Dissociation constant (K D In some embodiments, the labeled affinity reagent selectively binds to two or more types of amino acids with a K of less than about 100 nM, less than about 50 nM, less than about 25 nM, less than about 10 nM, or less than about 1 nM. D In some embodiments, the labeled affinity reagent selectively binds to two or more types of amino acids at a K of about 50 nM to about 50 μM (e.g., about 50 nM to about 500 nM, about 50 nM to about 5 μM, about 500 nM to about 50 μM, about 5 μM to about 50 μM, or about 10 μM to about 50 μM). D In some embodiments, the labeled affinity reagent selectively binds to two or more types of amino acids with a K D It selectively binds to two or more types of amino acids.

[0072] 1C illustrates various exemplary configurations and uses of labeled affinity reagents in accordance with the methods and compositions provided herein. In some embodiments, the labeled affinity reagent 100 includes a light-emitting label 110 (e.g., a label) and an affinity reagent (shown as a spotted shape) that selectively binds to one or more types of terminal amino acids of a polypeptide 120. In some embodiments, the affinity reagent is selective for one type of amino acid or a subset of types of amino acids (e.g., fewer than 20 common types of amino acids) at terminal positions or both terminal and internal positions.

[0073] As described herein, an affinity reagent (also known as a "recognition molecule") can be any biomolecule capable of selectively or specifically binding to one molecule over another (e.g., to one type of amino acid over another type of amino acid, similar to the "amino acid recognition molecules" referred to herein). In some embodiments, the affinity reagent is not a peptidase or does not have peptidase activity. For example, in some embodiments, the polypeptide sequencing method of the present application comprises contacting a polypeptide molecule with one or more affinity reagents and a cleavage reagent. In such embodiments, the one or more affinity reagents do not have peptidase activity, and removal of one or more amino acids from the polypeptide molecule (e.g., removal of an amino acid from a terminus of the polypeptide molecule) is performed by the cleavage reagent.

[0074] Affinity reagents (e.g., recognition molecules) include, for example, proteins and nucleic acids, which can be synthetic or recombinant. In some embodiments, the affinity reagent or recognition molecule can be an antibody or antigen-binding portion of an antibody, an SH2 domain-containing protein or fragment thereof, or an enzymatic biomolecule, such as a peptidase, aminotransferase, ribozyme, aptazyme, or tRNA synthetase (including the aminoacyl-tRNA synthetases and related molecules described in U.S. Patent Application Serial No. 15 / 255,433, filed September 2, 2016, entitled "MOLECULES AND METHODS FOR ITERATIVE POLYPEPTIDE ANALYSIS AND PROCESSING").

[0075] In some embodiments, the affinity reagent or recognition molecule of the present application is a degradation pathway protein. Examples of degradation pathway proteins suitable for use as recognition molecules include, but are not limited to, N-end rule pathway proteins, such as Arg / N-end rule pathway proteins, Ac / N-end rule pathway proteins, and Pro / N-end rule pathway proteins. In some embodiments, the recognition molecule is an N-end rule pathway protein selected from Gid proteins (e.g., Gid4 or Gid10 proteins), UBR box proteins (e.g., UBR1, UBR2) or UBR box domain-containing protein fragments thereof, p62 proteins or ZZ domain-containing fragments thereof, and ClpS proteins (e.g., ClpS1, ClpS2).

[0076] In some embodiments, the affinity reagent or recognition molecule of the present application is a ClpS protein, such as Agrobacterium tumifaciens ClpS1, Agrobacterium tumifaciens ClpS2, Synechococcus elongatus ClpS1, Synechococcus elongatus ClpS2, Thermosynechococcus elongatus ClpS, Escherichia coli ClpS, or Plasmodium falciparum ClpS. In some embodiments, the recognition molecule is an L / F transferase, such as Escherichia coli leucyl / phenylalanyl tRNA protein transferase. In some embodiments, the recognition molecule is a D / E leucyl transferase, such as Vibrio vulnificus aspartate / glutamate leucyl transferase Bpt. In some embodiments, the recognition molecule is a UBR protein or a UBR box domain, such as a UBR protein or the UBR box domain of human UBR1 and UBR2 or Saccharomyces cerevisiae UBR1. In some embodiments, the recognition molecule is a p62 protein, such as the H. sapiens p62 protein or the Rattus norvegicus p62 protein or a truncation variant thereof that contains at least the ZZ domain. In some embodiments, the recognition molecule is a Gid4 protein, such as H. sapiens GID4 or Saccharomyces cerevisiae GID4.In some embodiments, the recognition molecule is a Gid10 protein, e.g., Saccharomyces cerevisiae GID10. In some embodiments, the recognition molecule is an N-meristoyltransferase, e.g., Leishmania major N-meristoyltransferase or H. sapiens N-meristoyltransferase NMT1. In some embodiments, the recognition molecule is a BIR2 protein, e.g., Drosophila melanogaster BIR2. In some embodiments, the recognition molecule is a tyrosine kinase or an SH2 domain of a tyrosine kinase, e.g., the H. sapiens Fyn SH2 domain, the H. sapiens Src tyrosine kinase SH2 domain, or a variant thereof, e.g., the H. sapiens Fyn SH2 domain triple mutant superbinder. In some embodiments, the recognition molecule is an antibody or antibody fragment, e.g., a single chain antibody variable fragment (scFv) against phosphotyrosine or other post-translationally modified amino acid variants described herein.

[0077] Table 1 provides a list of exemplary sequences of amino acid recognition molecules. Also shown is the amino acid binding preference of each molecule relative to the amino acid identity of the terminal position of the polypeptide, unless otherwise specified in Table 1. It should be recognized that these sequences and other examples described herein are intended to be non-limiting, and that the recognition molecules of the present application can include any homologs, variants, or fragments thereof that contain, at a minimum, the domain or subdomain responsible for peptide recognition.

[0078] [Table 1-1]

[0079] [Table 1-2]

[0080] [Table 1-3]

[0081] [Table 1-4]

[0082] [Table 1-5]

[0083] [Table 1-6]

[0084] Thus, in some embodiments, the present application provides an amino acid recognition molecule having an amino acid sequence selected from Table 1 (or having an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, 80-90%, 90-95%, 95-99% or more amino acid sequence identity to an amino acid sequence selected from Table 1). In some embodiments, the amino acid recognition molecule has 25-50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-95%, or 95-99% or more amino acid sequence identity to an amino acid recognition molecule listed in Table 1. In some embodiments, the amino acid recognition molecule is a modified amino acid recognition molecule and includes one or more amino acid mutations relative to the sequence shown in Table 1.

[0085] In some embodiments, the amino acid recognition molecule comprises a tag sequence that provides one or more functions other than amino acid binding. For example, in some embodiments, the tag sequence comprises a biotin ligase recognition sequence that allows for biotinylation of the recognition molecule (e.g., incorporation of one or more biotin molecules, including biotin and bisbiotin moieties). Additional examples of functional sequences in the tag sequence include purification tags, cleavage sites, and other moieties useful for purifying and / or modifying the recognition molecule. Table 2 provides a non-limiting list of terminal tag sequences, any one or more of which may be used in combination with any one of the amino acid recognition molecules of the present application (e.g., in combination with the sequences shown in Table 1). It should be appreciated that the tag sequences shown in Table 2 are intended to be non-limiting, and that the recognition molecules of the present application may include any one or more of the tag sequences (e.g., His tags and / or biotinylation tags) at the N-terminus or C-terminus of the recognition molecule polypeptide, may be split between the N-terminus and C-terminus, or may be otherwise reconfigured as practiced in the art.

[0086] [Table 2]

[0087] In some embodiments, the recognition molecule or affinity reagent of the present application is a peptidase. Peptidases, also referred to as proteases or proteinases, are enzymes that catalyze the hydrolysis of peptide bonds. Peptidases can be generally classified as endopeptidases and exopeptidases, which digest polypeptides into shorter fragments and cleave the polypeptide chain internally or at the end, respectively. In some embodiments, the labeled affinity reagent 100 comprises a peptidase that has been modified to inactivate exopeptidase or endopeptidase activity. Thus, the labeled affinity reagent 100 selectively binds without cleaving amino acids from the polypeptide. In still other embodiments, a peptidase that has not been modified to inactivate exopeptidase or endopeptidase activity can be used. For example, in some embodiments, the labeled affinity reagent comprises a labeled exopeptidase 102.

[0088] According to certain embodiments of the present application, the protein sequencing method may include repeated detection and cleavage of the termini of a polypeptide. In some embodiments, a labeled exopeptidase 102 may be used as a single reagent that performs both amino acid detection and cleavage steps. As generally depicted, in some embodiments, the labeled exopeptidase 102 has aminopeptidase or carboxypeptidase activity to selectively bind and cleave amino acids at the N-terminus or C-terminus of a polypeptide, respectively. It should be appreciated that in certain embodiments, the labeled exopeptidase 102 may be catalytically inactivated by one skilled in the art to retain selective binding to use the labeled exopeptidase 102 as a non-cleavable labeled affinity reagent 100 as described herein.

[0089] Exopeptidases generally require a polypeptide substrate that contains at least one of a free amino group at its amino terminus or a free carboxyl group at its carboxy terminus. In some embodiments, the exopeptidases herein hydrolyze bonds at or near the termini of a polypeptide. In some embodiments, the exopeptidase hydrolyzes bonds three or fewer residues from the polypeptide terminus. For example, in some embodiments, a single hydrolysis reaction catalyzed by an exopeptidase cleaves a single amino acid, dipeptide, or tripeptide from the polypeptide terminus.

[0090] In some embodiments, the exopeptidases provided herein are aminopeptidases or carboxypeptidases that cleave single amino acids from the amino or carboxy terminus, respectively. In some embodiments, the exopeptidases provided herein are dipeptidyl peptidases or peptidyl dipeptidases that cleave dipeptides from the amino or carboxy terminus, respectively. In yet other embodiments, the exopeptidases provided herein are tripeptidyl peptidases that cleave tripeptides from the amino terminus. The classification and activity of peptidases of each class or subclass thereof are well known and described in the literature (see, for example, Gurupriya, VS and Roy, SC, Proteases and Protease Inhibitors in Male Reproduction, Proteases in Physiology and Pathology, pp. 195-216, 2017; and Brix, K. and Stocker, W., Proteases: Structure and Function, Chapter 1). In some embodiments, the peptidases of the present application remove more than three amino acids from a polypeptide terminus. Thus, in some embodiments, the peptidase is, for example, an endopeptidase that preferentially cleaves at a particular position (e.g., before or after a particular amino acid). In some embodiments, the size of the polypeptide cleavage product of the endopeptidase activity will depend on the distribution of cleavage sites (e.g., amino acids) within the polypeptide being analyzed.

[0091] The exopeptidase of the present application may be selected or engineered based on the directionality of the sequencing reaction. For example, in an embodiment of sequencing from the amino to carboxy terminus of a polypeptide, the exopeptidase comprises aminopeptidase activity. Conversely, in an embodiment of sequencing from the carboxy to amino terminus of a polypeptide, the exopeptidase comprises carboxypeptidase activity. Examples of carboxypeptidases that recognize specific carboxy terminal amino acids and can be used as labeled exopeptidases or inactivated for use as non-cleavable labeled affinity reagents as described herein are described in the literature (see, for example, Garcia-Guerrero, MC et al., 2018, Proceedings of the National Academy of Sciences of the United States of America (PNAS), Vol. 115, No. 17).

[0092] Peptidases suitable for use as cleavage reagents and / or affinity reagents (e.g., recognition molecules) include aminopeptidases that selectively bind to one or more types of amino acids. In some embodiments, the aminopeptidase recognition molecule is modified to inactivate aminopeptidase activity. In some embodiments, the aminopeptidase cleavage reagent is non-specific, cleaving most or all types of amino acids from the terminus of a polypeptide. In some embodiments, the aminopeptidase cleavage reagent cleaves one or more types of amino acids from the terminus of a polypeptide more efficiently than other types of amino acids at the terminus of the polypeptide. For example, the aminopeptidases of the present application specifically cleave alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, selenocysteine, serine, threonine, tryptophan, tyrosine, and / or valine. In some embodiments, the aminopeptidase is a proline aminopeptidase. In some embodiments, the aminopeptidase is a proline iminopeptidase. In some embodiments, the aminopeptidase is a glutamate / aspartate specific aminopeptidase. In some embodiments, the aminopeptidase is a methionine specific aminopeptidase. In some embodiments, the aminopeptidase is an aminopeptidase shown in Table 3. In some embodiments, the aminopeptidase cleavage reagent cleaves a peptide substrate shown in Table 3.

[0093] In some embodiments, the aminopeptidase is a non-specific aminopeptidase. In some embodiments, the non-specific aminopeptidase is a zinc metalloprotease. In some embodiments, the non-specific aminopeptidase is an aminopeptidase shown in Table 4. In some embodiments, the non-specific aminopeptidase cleaves a peptide substrate shown in Table 4.

[0094] Thus, in some embodiments, the present application provides an aminopeptidase (e.g., aminopeptidase recognition molecule, aminopeptidase cleavage reagent) having an amino acid sequence selected from Table 3 or Table 4 (or having an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, 80-90%, 90-95%, 95-99%, or more amino acid sequence identity to an amino acid sequence selected from Table 3 or Table 4). In some embodiments, the aminopeptidase has 25-50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-95%, or 95-99%, or more amino acid sequence identity to an aminopeptidase listed in Table 3 or Table 4. In some embodiments, the aminopeptidase is a modified aminopeptidase and includes one or more amino acid mutations relative to the sequence shown in Table 3 or Table 4.

[0095] [Table 3-1]

[0096] [Table 3-2]

[0097] [Table 4-1]

[0098] [Table 4-2]

[0099] [Table 4-3]

[0100] [Table 4-4]

[0101] [Table 4-5]

[0102] For purposes of comparing two or more amino acid sequences, the percentage of "sequence identity" (also referred to herein as "amino acid identity") between a first amino acid sequence and a second amino acid sequence may be calculated by dividing the number of amino acid residues in the first amino acid sequence that are identical to the amino acid residues at the corresponding positions in the second amino acid sequence by the total number of amino acid residues in the first amino acid sequence and multiplying by 0100. In this case, each deletion, insertion, substitution, or addition of an amino acid residue in the second amino acid sequence compared to the first amino acid sequence is considered to be the difference of a single amino acid residue (position). Alternatively, the degree of sequence identity between two amino acid sequences may be calculated using known computer algorithms (e.g., by the local homology algorithm of Smith and Waterman, 1970, Adv. Appl. Math., vol. 2, p. 482c; Needleman and Wunsch, J. Mol. Biol., 1970, pp. 111-115, 2002; and by the local homology algorithm of Smith and Waterman, 1970, Adv. Appl. Math., vol. 2, p. 482c; Needleman and Wunsch, J. Mol. Biol., 1970, pp. 111-115, 2002). 48, 443, by the similarity search method of Pearson and Lipman, Proc. Natl. Acad. Sci. USA, 1998, 85, 2444, or by using algorithms available as Blast, Clustal, Omega, or other sequence alignment algorithms implemented on a computer, e.g., with standard settings. Typically, for purposes of determining the percentage of "sequence identity" between two amino acid sequences according to the calculation methods outlined above, the amino acid sequence having the greatest number of amino acid residues will be considered the "first" amino acid sequence and the other amino acid sequence will be considered the "second" amino acid sequence.

[0103] Additionally or alternatively, two or more sequences may be evaluated for the identity between the sequences. The term "identical" or percent "identity" in the context of two or more nucleic acid or amino acid sequences refers to two or more sequences or subsequences that are identical. Two sequences are "substantially identical" if they have a certain percentage of amino acid residues or nucleotides that are identical (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical) over a designated region or over the entire sequence when aligned for maximum correspondence over a comparison window or designated region measured using one of the sequence comparison algorithms described above or by manual alignment and visual inspection. Optionally, the identity exists over a region that is at least about 25, 50, 75, or 100 amino acids in length, or over a region that is 100-150, 150-200, 100-200, or over 200 or more amino acids in length.

[0104] Additionally or alternatively, two or more sequences may be evaluated for alignment between the sequences. The term "alignment" or percent "alignment" in the context of two or more nucleic acid or amino acid sequences refers to two or more sequences or subsequences that are identical. Two sequences are "substantially aligned" if they have a certain percentage of amino acid residues or nucleotides that are identical (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical) over a designated region or over the entire sequence when aligned for maximum correspondence over a comparison window or designated region measured using one of the sequence comparison algorithms described above or by manual alignment and visual inspection. Optionally, the alignment occurs over a region that is at least about 25, 50, 75, or 100 amino acids in length, or over a region that is 100-150, 150-200, 100-200, or 200 or more amino acids in length.

[0105] In addition to protein molecules, nucleic acid molecules have a variety of advantageous properties for use as affinity reagents (eg, amino acid recognition molecules) in the present application. Nucleic acid aptamers are nucleic acid molecules engineered to bind to a desired target with high affinity and selectivity. Thus, nucleic acid aptamers can be engineered to selectively bind to a desired type of amino acid using selection and / or enrichment techniques known in the art. Thus, in some embodiments, the affinity reagent comprises a nucleic acid aptamer (e.g., DNA aptamer, RNA aptamer). As shown in FIG. 1C, in some embodiments, the labeled affinity reagent is a labeled aptamer 104 that selectively binds to one type of terminal amino acid. For example, in some embodiments, the labeled aptamer 104 selectively binds to one type of amino acid (e.g., a single type of amino acid or a subset of types of amino acids) at the terminus of a polypeptide, as described herein. Although not shown, it should be appreciated that the labeled aptamer 104 can be engineered to selectively bind to one type of amino acid at any position of a polypeptide (e.g., a terminal position or a terminal position and an internal position of a polypeptide) according to the methods of the present application.

[0106] In some embodiments, the labeled affinity reagent comprises a label having binding-induced emission. For example, in some embodiments, the labeled aptamer 106 comprises a donor label 112 and an acceptor label 114 and functions as illustrated in panels (I) and (II) of FIG. 1C. As depicted in panel (I), the labeled aptamer 106 as a free molecule assumes a conformation in which the donor label 112 and the acceptor label 114 are separated by a distance (e.g., about 10 nm or more) that limits detectable FRET between the labels. As depicted in panel (II), the labeled aptamer 106 as a selective binding molecule assumes a conformation in which the donor label 112 and the acceptor label 114 are within a distance (e.g., about 10 nm or less) that promotes detectable FRET between the labels. In yet other embodiments, the labeled aptamer 106 includes a quenching moiety and functions similarly to a molecular beacon, where the emission of the labeled aptamer 106 is internally quenched as a free molecule and restored as a selectively bound molecule (see, e.g., Hamaguchi et al., 2001, Analytical Biochemistry 294:126-131). Without wishing to be bound by theory, it is believed that these and other types of mechanisms of binding-induced emission advantageously reduce or eliminate background emission, increasing the overall sensitivity and accuracy of the methods described herein.

[0107] In addition to methods for identifying terminal amino acids of a polypeptide, the present application provides methods for sequencing a polypeptide using a labeled affinity reagent. In some embodiments, the sequencing method can include subjecting the polypeptide termini to repeated cycles of terminal amino acid detection and terminal amino acid cleavage. For example, in some embodiments, the present application provides methods for determining the amino acid sequence of a polypeptide, comprising contacting the polypeptide with one or more labeled affinity reagents described herein and subjecting the polypeptide to Edman degradation.

[0108] Traditional Edman degradation involves repeated cycles of modification and cleavage of the terminal amino acids of a polypeptide, identifying each successively cleaved amino acid to determine the amino acid sequence of the polypeptide. In an illustrative example of traditional Edman degradation, the N-terminal amino acid of a polypeptide is modified with phenylisothiocyanate (PITC) to form a PITC-derivatized N-terminal amino acid. The PITC-derivatized N-terminal amino acid is then cleaved using acidic conditions, basic conditions, and / or elevated temperatures. It has also been shown that the step of cleaving the PITC-derivatized N-terminal amino acid can be accomplished enzymatically, including using a modified cysteine ​​protease from the protozoan Trypanosoma cruzi and relatively milder cleavage conditions of neutral or near-neutral pH. Non-limiting examples of useful enzymes are described in U.S. patent application Ser. No. 15 / 255,433, filed Sep. 2, 2016, entitled "MOLECULES AND METHODS FOR ITERATIVE POLYPEPTIDE ANALYSIS AND PROCESSING."

[0109] An example of sequencing by Edman degradation using a labeled affinity reagent according to the present application is depicted in FIG. 1D. In some embodiments, sequencing by Edman degradation involves providing a polypeptide 122 immobilized to a surface 130 of a solid support (e.g., immobilized to the surface of the bottom or sidewall of a sample well) via a linker 124. In some embodiments, as described herein, the polypeptide 122 is immobilized at one end (e.g., the amino-terminal amino acid or the carboxy-terminal amino acid) such that the other end is free for detection and cleavage of the terminal amino acid. Thus, in some embodiments, the reagents used in the Edman degradation methods described herein preferentially interact with the terminal amino acid at the non-immobilized (e.g., free) end of the polypeptide 122. Thus, the polypeptide 122 remains immobilized over repeated cycles of detection and cleavage. To this end, in some embodiments, the linker 124 can be designed according to a desired set of conditions to be used for detection and cleavage, e.g., to limit detachment of the polypeptide 122 from the surface 130 under chemical cleavage conditions. Linker compositions and techniques suitable for immobilizing polypeptides to surfaces are described in detail elsewhere herein.

[0110] According to the present application, in some embodiments, the method of sequencing by Edman degradation includes step (1) of contacting a polypeptide 122 with one or more labeled affinity reagents that selectively bind to one or more types of terminal amino acids. As shown, in some embodiments, the labeled affinity reagent 108 interacts with the polypeptide 122 by selectively binding to the terminal amino acids. In some embodiments, step (1) further includes removing any of the one or more labeled affinity reagents that do not selectively bind to the terminal amino acids (e.g., the free terminal amino acids) of the polypeptide 122.

[0111] In some embodiments, the method further includes identifying the terminal amino acid of the polypeptide 122 by detecting the labeled affinity reagent 108. In some embodiments, the detecting includes detecting luminescence from the labeled affinity reagent 108. As described herein, in some embodiments, the luminescence is uniquely associated with the labeled affinity reagent 108, such that the luminescence is associated with the type of amino acid to which the labeled affinity reagent 108 selectively binds. Thus, in some embodiments, the type of amino acid is identified by determining one or more luminescent properties of the labeled affinity reagent 108.

[0112] In some embodiments, the Edman degradation sequencing method includes a step (2) of removing a terminal amino acid of the polypeptide 122. In some embodiments, step (2) includes removing a labeled affinity reagent 108 (e.g., any of one or more labeled affinity reagents that selectively bind to the terminal amino acid) from the polypeptide 122. In some embodiments, step (2) includes modifying a terminal amino acid (e.g., a free terminal amino acid) of the polypeptide 122 by contacting the terminal amino acid with an isothiocyanate (e.g., PITC) to form an isothiocyanate-modified terminal amino acid. In some embodiments, the isothiocyanate-modified terminal amino acid is more susceptible to removal by a cleavage reagent (e.g., a chemical or enzymatic cleavage reagent) than the unmodified terminal amino acid.

[0113] In some embodiments, step (2) comprises removing the terminal amino acid by contacting the polypeptide 122 with a protease 140 that specifically binds to and cleaves the isothiocyanate-modified terminal amino acid. In some embodiments, the protease 140 comprises a modified cysteine ​​protease. In some embodiments, the protease 140 comprises a modified cysteine ​​protease, such as a cysteine ​​protease from Trypanosoma cruzi (see, e.g., Borgo et al., 2015, Protein Science 24:571-579). In still other embodiments, step (2) comprises removing the terminal amino acid by subjecting the polypeptide 122 to chemical conditions (e.g., basic, acidic) sufficient to cleave the isothiocyanate-modified terminal amino acid.

[0114] In some embodiments, the method of sequencing by Edman degradation includes a step (3) of washing the polypeptide 122 after terminal amino acid cleavage. In some embodiments, the washing includes removing the protease 140. In some embodiments, the washing includes restoring neutral pH conditions to the polypeptide 122 (e.g., after chemical cleavage by acidic or basic conditions). In some embodiments, the method of sequencing by Edman degradation includes repeating steps (1)-(3) for multiple cycles.

[0115] An example method for sequencing by Edman degradation is shown in FIG. 1E. In some embodiments, a sample containing a complex mixture of polypeptides (e.g., a mixture of proteins) can be degraded into short polypeptide fragments of approximately 6-40 amino acids using common enzymes. In some embodiments, sequencing of this polypeptide library according to the methods of the present application will reveal the identity and abundance of each of the polypeptides present in the original complex mixture. As described herein and in the literature, most polypeptides within the size range of 6-40 amino acids can be uniquely identified by determining the number and position of just four amino acids in the polypeptide chain.

[0116] Thus, in some embodiments, Edman degradation sequencing methods can be performed using a set of labeled aptamers 150 that includes four DNA aptamer types, each type recognizing a different N-terminal amino acid. Each aptamer type can be labeled with a different luminescent label so that the different aptamer types can be distinguished based on one or more luminescent properties. For illustrative purposes, an example set of labeled aptamers 150 includes a cysteine-specific aptamer labeled with a first luminescent label ("dye 1"), a lysine-specific aptamer labeled with a second luminescent label ("dye 2"), a tryptophan-specific aptamer labeled with a third luminescent label ("dye 3"), and a glutamate-specific aptamer labeled with a fourth luminescent label ("dye 4").

[0117] In some embodiments, the Edman degradation sequencing method of the present application proceeds according to process 152 shown in FIG. 1E. In some embodiments, prior to step (1), single polypeptide molecules from a polypeptide library are immobilized on a surface of a solid support, for example, on the surface of the bottom or sidewall of a sample well of an array of sample wells. In some embodiments, as described elsewhere herein, moieties that enable surface immobilization (e.g., biotin) or improve solubility (e.g., oligonucleotides) can be chemically or enzymatically attached to the C-terminus of the polypeptide. To determine the sequence of each polypeptide, in some embodiments, the immobilized polypeptide is subjected to repeated cycles of N-terminal amino acid detection and N-terminal amino acid cleavage, as exemplified by process 152. In some embodiments, process 152 includes steps of reagent addition and washing, which are performed by injection onto the detection surface of a flow cell using an automated fluidic system. In some embodiments, steps (1)-(4) exemplify one cycle of detection and cleavage using a labeled aptamer 150.

[0118] In some embodiments, the Edman degradation sequencing method according to process 152 includes step (1) of incubating in a mixture of four orthogonally labeled DNA aptamers to allow binding of the aptamers to any immobilized polypeptides (e.g., polypeptides immobilized in sample wells of an array) that contain one of the four correct amino acids at their N-terminus. In some embodiments, the method further includes washing the immobilized polypeptides to remove unbound aptamers. In some embodiments, the method further includes imaging the immobilized polypeptides ("imaging step 1"). In some embodiments, the acquired image contains sufficient information to determine the location of the aptamer-binding polypeptides (e.g., locations in an array of sample wells) and which of the four aptamers are bound to each location. In some embodiments, the method further includes washing the immobilized polypeptides with a suitable buffer to remove the aptamers from the immobilized polypeptides.

[0119] In some embodiments, the sequencing method according to process 152 includes a step (2) of flowing in a solution containing a reactive molecule (e.g., PITC as shown) that specifically modifies the N-terminal amine group. An isothiocyanate molecule such as PITC modifies the N-terminal amino acid in some embodiments such that it becomes a substrate for cleavage by a modifying protease, such as the cysteine ​​protease cruzain from Trypanosoma Cruzi.

[0120] In some embodiments, the sequencing method according to process 152 includes step (3) of washing the immobilized polypeptide before flowing in a suitable modified protease that recognizes the modified N-terminal amino acid and cleaves it from the immobilized polypeptide. In some embodiments, the method includes step (4) of washing the immobilized polypeptide after enzymatic cleavage. In some embodiments, steps (1)-(4) depict one cycle of Edman degradation. Thus, step (1') shown is the start of the next reaction cycle which proceeds as steps (1')-(4') performed as described above for steps (1)-(4). In some embodiments, steps (1)-(4) are repeated for approximately 20-40 cycles.

[0121] In some embodiments, a labeled isothiocyanate (e.g., dye-labeled PITC) may be used to monitor sample loading. For example, in some embodiments, before subjecting a polypeptide sample to the sequencing method shown in process 152, the polypeptide sample is preconjugated at its end with a luminescent label by end modification using dye-labeled PITC. Thus, loading of the polypeptide sample into the array of sample wells may be monitored by detecting emission from the label before the start of process 152. In some embodiments, the emission may be used to determine the single occupancy of a sample well in the array (e.g., a portion of a sample well containing a single polypeptide molecule), thereby advantageously increasing the amount of information reliably obtained for a given sample. Once the desired sample loading status has been determined by emission, process 152 may be initiated by chemical or enzymatic cleavage as described before proceeding with step (1).

[0122] In some embodiments, a labeled isothiocyanate (e.g., dye-labeled PITC) may be used to monitor the reaction progress of a polypeptide sample in an array. For example, in some embodiments, step (2) involves flowing in a solution containing dye-labeled PITC that specifically modifies and labels the N-terminal amine groups of polypeptides in the sample. In some embodiments, light emission from the label may be detected during or after step (2) of evaluating N-terminal PITC modification of polypeptides in the sample. Thus, in some embodiments, light emission is used to determine whether or when to proceed from step (2) to step (3). In some embodiments, light emission from the label may be detected during or after step (3) of evaluating N-terminal amino acid cleavage of polypeptides in the sample to determine whether or when to proceed from step (3) to step (4).

[0123] The sequencing method according to process 152 utilizes separate reagents for detection and cleavage of terminal amino acids of a polypeptide. Nevertheless, in some embodiments, the present application provides a sequencing method that may use a single reagent comprising a peptidase for detection and cleavage of terminal amino acids of a polypeptide. Figure 2 shows an example of polypeptide sequencing using a set of labeled exopeptidases 200, each of which selectively binds to and cleaves a different type of terminal amino acid.

[0124] As generally illustrated in the example of Figure 2, the labeled exopeptidases 200 include a lysine-specific exopeptidase with a first luminescent label, a glycine-specific exopeptidase with a second luminescent label, an aspartate-specific exopeptidase with a third luminescent label, and a leucine-specific exopeptidase with a fourth luminescent label. According to certain embodiments described herein, each of the labeled exopeptidases 200 selectively binds to and cleaves its respective amino acid only if that amino acid is at the amino or carboxy terminus of the polypeptide. Therefore, since sequencing by this approach proceeds from one end to the other, the labeled exopeptidases 200 are engineered or selected such that all reagents in the set have either aminopeptidase activity or carboxypeptidase activity.

[0125] As further shown in Figure 2, process 202 illustrates a schematic of a real-time sequencing reaction using a labeled exopeptidase 200. Panels (I)-(IX) illustrate a progression of events involving repeated detection and cleavage at the ends of a polypeptide in conjunction with the signal output shown below corresponding to the events depicted in each panel. For purposes of illustration, a polypeptide having an arbitrarily selected amino acid sequence of "KLDG..." is shown (progressing from one end to the other).

[0126] Panel (I) depicts the start of a sequencing reaction where a polypeptide is immobilized on a surface of a solid support, such as the bottom or sidewall surface of a sample well. In some embodiments, the present sequencing methods include real-time single molecule sequencing. In some embodiments, multiple single molecule sequencing reactions are performed simultaneously in an array of sample wells. In such embodiments, polypeptide immobilization prevents diffusion of the polypeptide out of the sample well by anchoring the polypeptide within the sample well for single molecule analysis.

[0127] Panel (II) depicts a detection event in which a lysine-specific exopeptidase from the set of labeled affinity reagents 200 selectively binds to a terminal lysine residue of a polypeptide. As shown in the lower signal traces of panels (I) and (II), the signal output reports on this binding event by displaying an increase in signal intensity, and can be used to identify the terminal amino acid by identifying the luminescent label of the lysine-specific exopeptidase. Panel (III) illustrates that the labeled peptidase cleaves the terminal amino acid after selectively binding to it. As a result, this component is free to diffuse away from the observation region for luminescence detection, and is reported in the signal output by a decrease in signal intensity, as shown in the lower trace of panel (III). Panels (IV)-(IX) proceed through the process similarly as described for panels (I)-(III). That is, the labeled exopeptidase binds to and cleaves the corresponding terminal amino acid, generating a corresponding increase and decrease in signal output, respectively.

[0128] In some aspects, the present application provides a method for real-time polypeptide sequencing by evaluating the binding interaction of a terminal amino acid with a labeled amino acid recognition molecule (e.g., a labeled affinity reagent) and a labeled cleavage reagent (e.g., a labeled non-specific exopeptidase). FIG. 3A shows an example of a sequencing method in which discrete binding events generate signal pulses in a signal output 300. The inset panel of FIG. 3A illustrates a general scheme of real-time sequencing by this approach. As shown, a labeled affinity reagent 310 selectively associates (e.g., binds) with and dissociates from a terminal amino acid (shown here as lysine) to generate a series of pulses in the signal output 300 that can be used to identify the terminal amino acid. In some embodiments, the series of pulses provides a pulsing pattern (e.g., a characteristic pattern) that can be diagnostic of the identity of the corresponding terminal amino acid.

[0129] Without wishing to be bound by theory, the labeled affinity reagent 310 may be a ligand that regulates the association rate or "on" rate of binding (k on ) and the bond dissociation or "off" rate (k off ) and selectively bind according to the binding affinity (KD) defined by the rate constant k off and k on and are critical determinants of the pulse duration (e.g., the time corresponding to a detectable binding event) and inter-pulse duration (e.g., the time between detectable binding events), respectively. In some embodiments, these rates can be engineered to achieve the pulse duration and pulse rate (e.g., signal pulse frequency) that gives the best sequencing accuracy.

[0130] As shown in the inset panel, the sequencing reaction mixture further includes a labeled non-specific exopeptidase 320 that includes a luminescent label different from that of the labeled affinity reagent 310. In some embodiments, the labeled non-specific exopeptidase 320 is present in the mixture at a concentration less than that of the labeled affinity reagent 310. In some embodiments, the labeled non-specific exopeptidase 320 exhibits broad specificity such that it cleaves most or all types of terminal amino acids. Thus, a dynamic sequencing approach may include monitoring affinity reagent binding to the terminus of a polypeptide throughout the course of a degradation reaction catalyzed by the exopeptidase cleavage activity.

[0131] As illustrated by the progression of signal output 300, in some embodiments, terminal amino acid cleavage by labeled non-specific exopeptidase 320 generates signal pulses, and these events occur at a lower frequency than the binding pulses of labeled affinity reagent 310. Thus, amino acids of a polypeptide may be counted and / or identified in a real-time sequencing process. As further illustrated by signal output 300, in some embodiments, multiple labeled affinity reagents may be used, each having a diagnostic pulsing pattern (e.g., a characteristic pattern) that may be used to identify the corresponding terminal amino acid. For example, in some embodiments, different characteristic patterns (exemplified by each of lysine, phenylalanine, and glutamine in signal output 300) correspond to the association of more than one labeled affinity reagent with different types of terminal amino acids. As described herein, it should be appreciated that a single affinity reagent that associates with more than one type of amino acid may be used in accordance with the present application. Thus, in some embodiments, different characteristic patterns correspond to the association of one labeled affinity reagent with different types of terminal amino acids.

[0132] As described herein, the signal pulse information can be used to identify amino acids based on a characteristic pattern in a series of signal pulses. In some embodiments, the characteristic pattern includes a plurality of signal pulses, each signal pulse including a pulse duration. In some embodiments, the plurality of signal pulses can be characterized by a summary statistic (e.g., mean, median, time decay constant) of the distribution of pulse durations in the characteristic pattern. In some embodiments, the average pulse duration of the characteristic pattern is from about 1 millisecond to about 10 seconds (e.g., from about 1 ms to about 1 s, from about 1 ms to about 100 ms, from about 1 ms to about 10 ms, from about 10 ms to about 10 s, from about 100 ms to about 10 s, from about 1 s to about 10 s, from about 10 ms to about 100 ms, or from about 100 ms to about 500 ms). In some embodiments, different characteristic patterns corresponding to different types of amino acids in a single polypeptide can be distinguished from one another based on the statistical significance of the summary statistics. For example, in some embodiments, one characteristic pattern may be distinguishable from other characteristic patterns based on an average pulse duration difference of at least 10 milliseconds (e.g., about 10 ms to about 10 s, about 10 ms to about 1 s, about 10 ms to about 100 ms, about 100 ms to about 10 s, about 1 s to about 10 s, or about 100 ms to about 1 s). It should be appreciated that in some embodiments, the smaller the average pulse duration difference between different characteristic patterns, the more pulse durations may be required within each characteristic pattern to distinguish them from one another with statistical confidence.

[0133] As detailed above, the real-time sequencing process exemplified by Figure 3A may generally include cycles of terminal amino acid recognition and terminal amino acid cleavage, with the relative occurrence of recognition and cleavage being controllable by the concentration difference between the labeled affinity reagent 310 and the labeled non-specific exopeptidase 320. In some embodiments, the concentration difference can be optimized so that the number of signal pulses detected upon recognition of individual amino acids provides a desired confidence interval for identification. For example, if an initial sequencing reaction provides signal data with too few signal pulses between cleavage events to allow for the determination of a characteristic pattern with a desired confidence interval, the sequencing reaction can be repeated with a reduced concentration of the non-specific exopeptidase compared to the affinity reagent.

[0134] In some embodiments, polypeptide sequencing according to the present application can be performed by contacting the polypeptide with a sequencing reaction mixture comprising one or more amino acid recognition molecules (e.g., affinity reagents) and / or one or more cleavage reagents (e.g., exopeptidases). In some embodiments, the sequencing reaction mixture comprises an amino acid recognition molecule at a concentration of about 10 nM to about 10 μM. In some embodiments, the sequencing reaction mixture comprises a cleavage reagent at a concentration of about 500 nM to about 500 μM.

[0135] In some embodiments, the sequencing reaction mixture comprises an amino acid recognition molecule at a concentration of about 100 nM to about 10 μM, about 250 nM to about 10 μM, about 100 nM to about 1 μM, about 250 nM to about 1 μM, about 250 nM to about 750 nM, or about 500 nM to about 1 μM. In some embodiments, the sequencing reaction mixture comprises an amino acid recognition molecule at a concentration of about 100 nM, about 250 nM, about 500 nM, about 750 nM, or about 1 μM.

[0136] In some embodiments, the sequencing reaction mixture comprises a cleavage reagent at a concentration of about 500 nM to about 250 μM, about 500 nM to about 100 μM, about 1 μM to about 100 μM, about 500 nM to about 50 μM, about 1 μM to about 100 μM, about 10 μM to about 200 μM, or about 10 μM to about 100 μM. In some embodiments, the sequencing reaction mixture comprises a cleavage reagent at a concentration of 1 μM, about 5 μM, about 10 μM, about 30 μM, about 50 μM, about 70 μM, or about 100 μM.

[0137] In some embodiments, the sequencing reaction mixture comprises an amino acid recognition molecule at a concentration of about 10 nM to about 10 μM and a cleavage reagent at a concentration of about 500 nM to about 500 μM. In some embodiments, the sequencing reaction mixture comprises an amino acid recognition molecule at a concentration of about 100 nM to about 1 μM and a cleavage reagent at a concentration of about 1 μM to about 100 μM. In some embodiments, the sequencing reaction mixture comprises an amino acid recognition molecule at a concentration of about 250 nM to about 1 μM and a cleavage reagent at a concentration of about 10 μM to about 100 μM. In some embodiments, the sequencing reaction mixture comprises an amino acid recognition molecule at a concentration of about 500 nM and a cleavage reagent at a concentration of about 25 μM to about 75 μM.

[0138] In some embodiments, the sequencing reaction mixture comprises an amino acid recognition molecule and a cleavage reagent in a ratio of about 500:1, about 400:1, about 300:1, about 200:1, about 100:1, about 75:1, about 50:1, about 25:1, about 10:1, about 5:1, about 2:1, or about 1:1. In some embodiments, the sequencing reaction mixture comprises an amino acid recognition molecule and a cleavage reagent in a ratio of about 10:1 to about 200:1. In some embodiments, the sequencing reaction mixture comprises an amino acid recognition molecule and a cleavage reagent in a ratio of about 50:1 to about 150:1.

[0139] Although the example illustrated by FIG. 3A concerns a sequencing process using a labeled cleavage reagent, the sequencing process is not intended to be limited in this respect. As described elsewhere herein, the inventors have demonstrated single molecule sequencing using unlabeled cleavage reagents. In some embodiments, the approximate frequency at which the cleavage reagent removes successive terminal amino acids is known, for example, based on the known activity and / or concentration of the enzyme used. In some embodiments, terminal amino acid cleavage by the reagent is inferred, for example, based on a detected signal or lack of detected signal upon amino acid recognition. The inventors have recognized additional control techniques for real-time sequencing reactions that can be used in combination with or instead of the concentration difference approach described.

[0140] An example of a temperature-dependent real-time sequencing process is shown in FIG. 3B. Panels (I)-(III) illustrate a sequencing reaction that includes cycles of temperature-dependent terminal amino acid recognition and terminal amino acid cleavage. Each cycle of the sequencing reaction involves two temperature ranges: a first temperature range ("T") that is optimal for affinity reagent activity (e.g., promoting terminal amino acid recognition) over exopeptidase activity; 1 ") and a second temperature range ("T 2 The sequencing reaction is carried out over a first temperature range T 1 (initiates amino acid recognition) and the second temperature range T 2 (which initiates amino acid cleavage). Thus, the progression of the temperature-dependent sequencing process can be controlled by temperature and can be controlled between different temperature ranges (e.g., T 1 and T 2 Switching between the first temperature range T 1 The affinity reagent activity (e.g., binding affinity for amino acids (KD)) within a second temperature range T 2In some embodiments, the second temperature range T 2 The exopeptidase activity (e.g., rate of substrate conversion to cleavage products) within a first temperature range T 1 is increased by at least 2-fold, 10-fold, at least 25-fold, at least 50-fold, at least 100-fold, at least 1,000-fold, or more compared to

[0141] In some embodiments, the first temperature range T 1 is the second temperature range T 2 In some embodiments, the first temperature range T 1 In some embodiments, the second temperature range T 2 In some embodiments, the first temperature range T 1 is about 20° C. to about 40° C. (for example, about 30° C.), and the second temperature range T 2 is about 60° C. to about 100° C. (for example, approximately 80° C.).

[0142] In some embodiments, the first temperature range T 1 is the second temperature range T 2 In some embodiments, the first temperature range T 1 In some embodiments, the second temperature range T 2 In some embodiments, the first temperature range T 1 is about 60° C. to about 100° C. (for example, about 80° C.), and the second temperature range T 2 is about 20° C. to about 40° C. (for example, approximately 30° C.).

[0143] Panel (I) shows the first temperature range T 1 1 depicts a sequencing reaction mixture at a temperature within a first temperature range T 1 When the mixture is within a first temperature range T 1 Within the first affinity reagent, the labeled exopeptidase in the mixture is inactivated (e.g., denatured) to prevent amino acid cleavage upon recognition. In panel (I), the first affinity reagent is shown reversibly associated with a terminal lysine of the polypeptide, while the labeled exopeptidase (e.g., Pfu aminopeptidase I (Pfu API)) is shown denatured. In some embodiments, amino acid recognition is performed for a predetermined duration before initiating amino acid cleavage. In some embodiments, amino acid recognition is performed for a duration required to achieve a desired confidence interval for identification before initiating amino acid cleavage. After amino acid recognition, the reaction is continued by increasing the temperature of the mixture to a second temperature range T 2 Progress is made by making changes within.

[0144] Panel (II) shows a second temperature range, T, that is more optimal for exopeptidase activity than affinity reagent activity. 2 For illustrative purposes in this example, a second temperature range T 2 is the first temperature range T 1 Although it should be appreciated that the reagent activity may be optimized for any desired temperature range. Thus, progression from panel (I) to panel (II) is accomplished by increasing the reaction mixture temperature using a suitable heat source. When the reaction mixture is heated to a second temperature range T 2When the mixture reaches a temperature within a second temperature range T 2 Within the first temperature range T, the labeled affinity reagent in the mixture is inactivated (e.g., denatured) to prevent amino acid recognition upon cleavage. In panel (II), the labeled exopeptidase is shown to cleave terminal lysine residues while the labeled affinity reagent is denatured. In some embodiments, amino acid cleavage is performed for a predetermined duration before initiating recognition of sequential amino acids at the polypeptide terminus. In some embodiments, amino acid cleavage is performed for a duration required to detect cleavage before initiating recognition of sequential amino acids. After amino acid cleavage, the reaction is continued by increasing the temperature of the mixture to a first temperature range T 1 Progress is made by making changes within.

[0145] Panel (III) shows the reaction mixture temperature being reduced to a first temperature range T 1 Thus, in this example, progression from panel (II) to panel (III) depicts the beginning of the next cycle during which the sequencing reaction returns to within the first temperature range T 1 This can be accomplished by cooling (e.g., actively or passively) to room temperature. As shown, the labeled affinity reagent is renatured, including a second affinity reagent that reversibly associates with the terminal phenylalanine of the polypeptide, while the labeled exopeptidase is shown denatured. The sequencing reaction continues by further cycling between amino acid recognition and amino acid cleavage in a temperature-dependent manner, as illustrated by this example.

[0146] Therefore, a dynamic sequencing approach may include reaction cycling controlled by the level of protein activity or function of one or more proteins in the reaction mixture. It should be appreciated that the temperature-dependent polypeptide sequencing process depicted in FIG. 3B and described above may exemplify a general approach to polypeptide sequencing by controllable cycling of condition-dependent recognition and cleavage. For example, in some embodiments, the present application provides a luminescence-dependent sequencing process using luminescence-activated reagents. In some embodiments, the luminescence-dependent sequencing process includes cycles of luminescence-dependent amino acid recognition and cleavage. Each cycle of the sequencing reaction may be performed by exposing the sequencing reaction mixture to two different luminescence conditions, i.e., a first luminescence condition that is optimal for affinity reagent activity (e.g., promotes amino acid recognition) over exopeptidase activity and a second luminescence condition that is optimal for exopeptidase activity (e.g., promotes amino acid cleavage) over affinity reagent activity. The sequencing reaction proceeds by switching between exposing the reaction mixture to the first luminescence condition (to initiate amino acid recognition) and exposing the reaction mixture to the second luminescence condition (to initiate amino acid cleavage). By way of example and not limitation, in some embodiments, the two different lighting conditions include a first wavelength and a second wavelength.

[0147] In some aspects, the present application provides a real-time polypeptide sequencing method by evaluating the binding interactions of one or more labeled affinity reagents with terminal and internal amino acids, and the binding interactions of a labeled non-specific exopeptidase with terminal amino acids. FIG. 4 shows an example of a sequencing method in which the method described and illustrated in the approach of FIGS. 3A-3B has been modified by using a labeled affinity reagent 410 that selectively binds to and dissociates from one type of amino acid (shown here as lysine) at both terminal and internal positions (inset panel of FIG. 4). As described in the previous approach, selective binding produces a series of pulses in the signal output 400. However, in this approach, the series of pulses occurs at a rate determined by the number of types of amino acids throughout the polypeptide. Therefore, in some embodiments, the pulsing rate corresponding to a binding event will be diagnostic of the number of cognate amino acids present at that time in the polypeptide.

[0148] As with the previous approach, the labeled non-specific peptidase 420 will be present at a relatively lower concentration than the labeled affinity reagent 410, for example, to provide an optimal time window between cleavage events (insert panel of FIG. 4). Additionally, in certain embodiments, the unique identifiable luminescent label of the labeled non-specific peptidase 420 will indicate when a cleavage event has occurred. As the polypeptide undergoes repeated cleavage, the pulsing rate corresponding to binding by the labeled affinity reagent 410 will decrease stepwise whenever a terminal amino acid is cleaved by the labeled non-specific peptidase 420. This concept is illustrated by plot 402, which generally depicts the pulsing rate as a function of time, with cleavage events at times represented by arrows. Thus, in some embodiments, amino acids may be identified and thus the polypeptide sequenced by this approach based on pulsing patterns and / or pulsing rates occurring within the patterns detected between cleavage events.

[0149] In some embodiments, terminal polypeptide sequence information (e.g., determined as described herein) can be combined with polypeptide sequence information obtained from one or more other sources. For example, terminal polypeptide sequence information can be combined with internal polypeptide sequence information. In some embodiments, internal polypeptide sequence information can be obtained using one or more amino acid recognition molecules associated with internal amino acids, as described herein. Internal or other polypeptide sequence information can be obtained before or during the polypeptide degradation process. In some embodiments, sequence information obtained from these methods can be combined with polypeptide sequence information using other techniques, for example, sequence information obtained using one or more internal amino acid recognition molecules.

[0150] Shielded Recognition Molecules According to embodiments described herein, a single molecule polypeptide sequencing method can be performed by illuminating a surface-immobilized polypeptide with excitation light and detecting the emission generated by a label attached to an amino acid recognition molecule (e.g., a labeled affinity reagent). In some cases, radiative and / or non-radiative decay generated by the label can cause photodamage to the polypeptide. For example, Figure 5A illustrates an example sequencing reaction in which a recognition molecule is shown associated with a surface-immobilized polypeptide.

[0151] In the presence of excitation illumination, the labels can generate fluorescence via radiative decay resulting in a detectable association event. However, in some cases, the labels generate non-radiative decay that can result in the formation of reactive oxygen species 500. The reactive oxygen species 500 can ultimately damage the immobilized peptide, resulting in the reaction being terminated before the full sequence information of the polypeptide can be obtained. This photodamage can occur, for example, at exposed polypeptide termini (top open arrow), at internal locations (middle open arrow), or at surface linkers that attach the polypeptide to a surface (bottom open arrow).

[0152] The inventors have found that photodamage can be reduced and recognition time can be extended by incorporating a shielding element into the amino acid recognition molecule. Figure 5B illustrates an example of a sequencing reaction using a shield recognition molecule that includes a shield 502. The shield 502 forms a covalent or non-covalent linking group that provides an increased distance between the label and the polypeptide, thereby reducing the damaging effect from reactive oxygen species 500 due to free radical decay over the label-polypeptide separation distance. The shield 502 can also provide a steric barrier that shields the polypeptide from the label by absorbing damage from reactive oxygen species 500 and radiative and / or non-radiative decay.

[0153] Without wishing to be bound by theory, it is believed that a shield positioned between a recognition moiety and a labeling moiety can absorb, deflect, or otherwise block radiation and / or non-radiative decay emitted by the labeling moiety. In some embodiments, the shield prevents or limits the interaction of one or more labels (e.g., luminescent labels) with one or more amino acid recognition molecules. In some embodiments, the shield prevents or limits the interaction of one or more labels with one or more molecules associated with the amino acid recognition molecule (e.g., polypeptides associated with the recognition molecule). Thus, in some embodiments, the term shield can generally refer to a protective or shielding effect provided by any part of the linking group formed between the recognition moiety and the labeling moiety.

[0154] In some embodiments, the shield is attached to one or more amino acid recognition molecules (e.g., recognition moieties) and one or more labels (e.g., labeling moieties). In some embodiments, the recognition moieties and labeling moieties are attached to non-adjacent sites on the shield. For example, one or more amino acid recognition molecules can be attached to a first side of the shield and one or more labels can be attached to a second side of the shield, where the first and second sides of the shield are spaced apart from one another. In some embodiments, the attachment sites are approximately on opposite sides of the shield.

[0155] The distance between the site where the shield is attached to the recognition molecule and the site where the shield is attached to the label can be a linear measurement through the space or a non-linear measurement across the surface of the shield. The distance between the recognition molecule binding site and the label binding site on the shield can be measured by modeling the three-dimensional structure of the shield. In some embodiments, this distance can be at least 2 nm, at least 4 nm, at least 6 nm, at least 8 nm, at least 10 nm, at least 12 nm, at least 15 nm, at least 20 nm, at least 30 nm, at least 40 nm, or more. Alternatively, the relative positions of the recognition molecule and the label on the shield can be described by treating the structure of the shield as a quadric surface (e.g., an ellipsoid, an ellipsoid cylinder). In some embodiments, the recognition molecule binding site and the label binding site are separated by a distance of at least 1 / 8 the distance around the ellipsoid shape representing the shield. In some embodiments, the recognition molecule and the label are separated by a distance of at least 1 / 4 the distance around the ellipsoid shape representing the shield. In some embodiments, the recognition molecule and the label are separated by a distance of at least 1 / 3 the distance around the ellipsoid shape representing the shield. In some embodiments, the recognition molecule and the label are separated by a distance that is ½ the distance around the ellipsoid shape representing the shield.

[0156] The size of the shield should be such that the label cannot or is unlikely to directly contact the polypeptide when the amino acid recognition molecule is associated with the polypeptide. The size of the shield should also be such that the attached label is detectable when the amino acid recognition molecule is associated with the polypeptide. For example, the size should be such that it is within the illumination volume where the attached luminescent label is excited.

[0157] It should be appreciated that there are various parameters by which a practitioner can assess the shielding effect. In general, the effect of a shielding element can be assessed by performing a comparative assessment between a composition having a shielding element and a composition lacking the shielding element. For example, a shielding element can increase the recognition time of an amino acid recognition molecule. In some embodiments, recognition time refers to the length of time during which an association event between a recognition molecule and a polypeptide is observable during a polypeptide sequencing reaction described herein. In some embodiments, the recognition time is increased by about 10-25%, 25-50%, 50-75%, 75-100%, or more than 100%, e.g., about 2-fold, 3-fold, 4-fold, 5-fold, or more, compared to a polypeptide sequencing reaction performed under identical conditions except that the amino acid recognition molecule lacks a shielding element but is otherwise similar or identical. In some embodiments, the shielding elements are capable of increasing sequencing accuracy and / or sequence read length (e.g., by at least 5%, at least 10%, at least 15%, at least 25%, or more compared to sequencing reactions performed under comparative conditions described above).

[0158] Thus, in some aspects, the present application provides a shielded recognition molecule comprising at least one amino acid recognition molecule, at least one detectable label, and a shielding element (e.g., a "shield") that forms a covalent or non-covalent linking group between the recognition molecule and the label. In some embodiments, the shielding element is at least 2 nm, at least 5 nm, at least 10 nm, at least 12 nm, at least 15 nm, at least 20 nm, or more in length (e.g., in an aqueous solution). In some embodiments, the shielding element is about 2 nm to about 100 nm in length (e.g., about 2 nm to about 50 nm, about 10 nm to about 50 nm, about 20 nm to about 100 nm).

[0159] In some embodiments, the shield (e.g., shielding element) forms a covalent or non-covalent linking group between one or more amino acid recognition molecules (e.g., recognition moieties) and one or more labels (e.g., labeling moieties). As used herein, in some embodiments, covalent and non-covalent linkages or linking groups refer to the nature of attachment of the recognition moieties and labeling moieties to the shield.

[0160] In some embodiments, covalent linkage or covalent linking group refers to a shield that is attached to each of the recognition moiety and labeling moiety via a covalent bond or a series of contiguous covalent bonds. Covalent attachment of one or both moieties can be achieved by covalent conjugation methods known in the art. For example, in some embodiments, click chemistry techniques (e.g., copper-catalyzed, strain-promoted, copper-free click chemistry, etc.) can be used to attach one or both moieties to the shield. Such methods generally involve conjugating one reactive moiety to the other reactive moiety to form one or more covalent bonds between the reactive moieties. Thus, in some embodiments, a first reactive moiety of the shield is contacted with a second reactive moiety of the recognition moiety or labeling moiety to form a covalent attachment. Examples of reactive moieties include, but are not limited to, reactive amines, azides, alkynes, nitrones, alkenes (e.g., cycloalkenes), tetrazines, tetrazoles, and other reactive moieties suitable for click reactions and similar coupling techniques.

[0161] In some embodiments, non-covalent linkage or non-covalent linking group refers to a shield attached to one or both of the recognition component and the label component via one or more non-covalent coupling means, such as, but not limited to, receptor-ligand interactions and oligonucleotide strand hybridization. Examples of receptor-ligand interactions are provided herein and include, but are not limited to, protein-protein complexes, protein-ligand complexes, protein-aptamer complexes, and aptamer-nucleic acid complexes. Various configurations and strategies for oligonucleotide strand hybridization are described herein and are known in the art (see, for example, US Patent Publication No. 2019 / 0024168).

[0162] In some embodiments, the shield 502 comprises a polymer, such as a biomolecule or a dendritic polymer. FIG. 5C shows an example of a polymer shield and the configuration of the shield recognition molecule of the present application. The first shield construct 504 shows an example of a protein shield 530. In some embodiments, the protein shield 530 forms a covalent linking group between the recognition molecule and the label. For example, in some embodiments, the protein shield 530 is attached to each of the recognition molecule and the label via one or more covalent bonds, for example, by covalent attachment via a side chain of a natural or unnatural amino acid of the protein shield 530. In some embodiments, the protein shield 530 forms a non-covalent linking group between the recognition molecule and the label. For example, in some embodiments, the protein shield 530 is a monomeric or multimeric protein that includes one or more ligand binding sites. In some embodiments, the non-covalent linking group is formed via one or more ligand moieties bound to one or more ligand binding sites. Additional examples of non-covalent linkages formed by protein shields are described elsewhere herein.

[0163] The second shield construct 506 shows an example of a double-stranded nucleic acid shield comprising a first oligonucleotide strand 532 hybridized to a second oligonucleotide strand 534. As shown, in some embodiments, the double-stranded nucleic acid shield can comprise a recognition molecule attached to the first oligonucleotide strand 532 and a label attached to the second oligonucleotide strand 534. Thus, the double-stranded nucleic acid shield forms a non-covalent linkage between the recognition molecule and the label via oligonucleotide strand hybridization. In some embodiments, the recognition molecule and the label can be attached to the same oligonucleotide strand, which can provide a double-stranded nucleic acid shield via hybridization with a single-stranded nucleic acid shield or another oligonucleotide strand. In some embodiments, strand hybridization can provide increased rigidity within the linkage to further enhance separation between the recognition molecule and the label.

[0164] When the shielding element 502 comprises a nucleic acid, the separation distance between the label and the recognition molecule can be measured by the distance between attachment sites on the nucleic acid (e.g., direct attachment or indirect attachment, such as via one or more additional shielding polymers). In some embodiments, the distance between attachment sites on the nucleic acid can be measured by the number of nucleotides in the nucleic acid that are between the label and the recognition molecule. It should be understood that the number of nucleotides can mean either the number of nucleotide bases in a single stranded nucleic acid or the number of nucleotide base pairs in a double stranded nucleic acid.

[0165] Thus, in some embodiments, the attachment site for the recognition molecule and the attachment site for the label can be separated by 5-200 nucleotides (e.g., 5-150 nucleotides, 5-100 nucleotides, 5-50 nucleotides, 10-100 nucleotides). It should be appreciated that any position in the nucleic acid can serve as an attachment site for a recognition molecule, a label, or one or more additional polymer shields. In some embodiments, the attachment site can be at or approximately the 5' or 3' end or an internal position along the strand of the nucleic acid.

[0166] The non-limiting configuration of the second shield construct 506 illustrates an example of a shield that forms a non-covalent linkage via strand hybridization. A further example of a non-covalent linkage is illustrated by the third shield construct 508, which includes an oligonucleotide shield 536. In some embodiments, the oligonucleotide shield 536 is a nucleic acid aptamer that binds to a recognition molecule to form a non-covalent linkage. In some embodiments, the recognition molecule is a nucleic acid aptamer, and the oligonucleotide shield 536 includes an oligonucleotide strand that hybridizes to the aptamer to form a non-covalent linkage.

[0167] The fourth shield construction 510 shows an example of a dendritic polymer shield 538. As used herein, in some embodiments, dendritic polymer generally refers to a polyol or a dendrimer. Polyols and dendrimers are described in the art and may include branched dendritic structures optimized for specific configurations. In some embodiments, the dendritic polymer shield 538 includes polyethylene glycol, tetraethylene glycol, poly(amidoamine), poly(propyleneimine), poly(propyleneamine), carbosilane, poly(L-lysine), or one or more combinations thereof.

[0168] Dendrimers or dendrons are repeatedly branched molecules that are typically symmetrical around a core and can assume a spherical three-dimensional shape. See, for example, Astruc et al., 2010, Chem.Rev., vol. 110, p. 1857. The incorporation of such structures into the shields of the present application can provide a protective effect through steric inhibition of contact between the label and one or more biomolecules associated therewith (e.g., recognition molecules and / or polypeptides associated therewith). Refinement of the chemical and physical properties of the dendrimer through variation of the primary structure of the molecule allows for tailoring of the shielding effect as desired, including potential functionalization of the dendrimer surface. Dendrimers can be synthesized by a variety of techniques using a wide range of materials and branching reactions known in the art. Such synthetic variations allow customization of the dendrimer properties as needed. Examples of polyol and dendrimer compounds that can be used in accordance with the SHIELD of the present application include, but are not limited to, those compounds described in U.S. Patent Application Publication No. 20180346507.

[0169] FIG. 5D shows further example configurations of shield recognition molecules of the present application. Protein-nucleic acid construct 512 shows an example of a shield comprising more than one polymer in the form of a protein and a double-stranded nucleic acid. In some embodiments, the protein portion of the shield is attached to the nucleic acid portion of the shield via a covalent linkage. In some embodiments, the attachment is via a non-covalent linkage. For example, in some embodiments, the protein portion of the shield is a monovalent or multivalent protein that forms at least one non-covalent linkage via a ligand portion attached to a ligand binding site of the monovalent or multivalent protein. In some embodiments, the protein portion of the shield comprises an avidin protein.

[0170] In some embodiments, the shield recognition molecule of the present application is an avidin nucleic acid construct 514. In some embodiments, the avidin nucleic acid construct 514 comprises a shield comprising an avidin protein 540 and a double stranded nucleic acid. As described herein, the avidin protein 540 can be used to form a non-covalent linkage between one or more amino acid recognition molecules and one or more labels, either directly or indirectly, such as through one or more additional shield polymers as described herein.

[0171] Avidin proteins are biotin-binding proteins, and generally have a biotin-binding site on each of the four subunits of the avidin protein. Avidin proteins include, for example, avidin, streptavidin, traptavidin, tamavidin, bladdervidin, xenavidin, and homologs and variants thereof. In some cases, monomeric, dimeric, or tetrameric forms of avidin proteins can be used. In some embodiments, the avidin protein of the avidin protein complex is a tetrameric form (e.g., homotetramer) of streptavidin. In some embodiments, the biotin-binding site of the avidin protein provides an attachment site for one or more amino acid recognition molecules, one or more labels, and / or one or more additional shielding polymers described herein.

[0172] An exemplary diagram of an avidin protein complex is shown in the inset panel of FIG. 5D. As shown in the inset panel, the avidin protein 540 can include binding sites 542 on each of the four subunits of the protein that can bind to a biotin moiety (shown as an open circle). The multivalency of the avidin protein 540 can allow for a variety of linking configurations, shown generally for illustrative purposes. For example, in some embodiments, a biotin linking moiety 544 can be used to provide a single attachment point to the avidin protein 540. In some embodiments, a bis-biotin linking moiety 546 can be used to provide two attachment points to the avidin protein 540. As exemplified by the avidin nucleic acid construct 514, an avidin protein complex can be formed with two bis-biotin linking moieties forming a trans configuration to provide an increased separation distance between the recognition molecule and the label.

[0173] Various further examples of avidin protein shield configurations are shown. The first avidin construct 516 shows an example of an avidin shield attached to a recognition molecule via a bis-biotin linking moiety and to two labels via separate biotin linking moieties. The second avidin construct 518 shows an example of an avidin shield attached to two recognition molecules via separate biotin linking moieties and to a label via a bis-biotin linking moiety. The third avidin construct 520 shows an example of an avidin shield attached to two recognition molecules via separate biotin linking moieties and to a label nucleic acid via a biotin linking moiety on each strand of the nucleic acid. The fourth avidin construct 522 shows an example of an avidin shield attached to a recognition molecule and to a label nucleic acid via separate bis-biotin linking moieties. As shown, the label is further shielded from the recognition molecule by a dendritic polymer between the label and the nucleic acid.

[0174] It should be appreciated that the example shield recognition molecule configurations shown in Figures 5A-5D are provided for illustrative purposes. The inventors have envisioned a variety of other shield configurations using one or more different polymers that form covalent or non-covalent links between the recognition moieties of the shield recognition molecules and the labeling moieties. As an example, Figure 5E illustrates the modularity of the shield configurations of the present application.

[0175] As shown at the top of FIG. 5E, a shielding recognition molecule generally includes a recognition component 550, a shielding element 552, and a label component 554. For ease of illustration, the recognition component 550 is depicted as one amino acid recognition molecule, and the label component 554 is depicted as one label. It should be appreciated that a shielding recognition molecule of the present application may include a shielding element 552 attached to one or more amino acid recognition molecules and one or more labels. When the recognition component 550 includes more than one recognition molecule, each recognition molecule can be attached to the shielding element 552 at one or more attachment sites on the shielding element 552. When the label component 554 includes more than one label, each label can be attached to the shielding element 552 at one or more attachment sites on the shielding element 552.

[0176] In some embodiments, the shielding element 552 comprises a protein 560. In some embodiments, the protein 560 is a monovalent or multivalent protein. In some embodiments, the protein 560 is a monomeric or multimeric protein, such as a protein homodimer, a protein heterodimer, a protein oligomer, or other protein molecule. In some embodiments, the shielding element 552 comprises a protein complex formed by a protein non-covalently bound to at least one other molecule. For example, in some embodiments, the shielding element 552 comprises a protein-protein complex 562. In some embodiments, the protein-protein complex 562 comprises one protein molecule specifically bound to another protein molecule. In some embodiments, the protein-protein complex 562 comprises an antibody or antibody fragment (e.g., scFv) bound to an antigen. In some embodiments, the protein-protein complex 562 comprises a receptor bound to a protein ligand. Additional examples of protein-protein complexes include, but are not limited to, trypsin-aprotinin, barnase-barstar, and colicin E9-Im9 immunity proteins.

[0177] In some embodiments, the shielding element 552 comprises a protein-ligand complex 564. In some embodiments, the protein-ligand complex 564 comprises a monovalent protein and a non-protein ligand moiety. For example, in some embodiments, the protein-ligand complex 564 comprises an enzyme bound to a small molecule inhibitor moiety. In some embodiments, the protein-ligand complex 564 comprises a receptor bound to a non-protein ligand moiety.

[0178] In some embodiments, shielding element 552 comprises a multivalent protein complex formed by a multivalent protein non-covalently bound to one or more ligand moieties. In some embodiments, shielding element 552 comprises an avidin protein complex formed by an avidin protein non-covalently bound to one or more biotin linking moieties. Constructs 566, 568, 570, and 572 provide illustrative examples of avidin protein complexes, any one or more of which may be incorporated into shielding element 552.

[0179] In some embodiments, the shielding element 552 comprises a two-way avidin complex 566 that includes an avidin protein bound to two bis-biotin linking moieties. In some embodiments, the shielding element 552 comprises a three-way avidin complex 568 that includes an avidin protein bound to two biotin linking moieties and a bis-biotin linking moiety. In some embodiments, the shielding element 552 comprises a four-way avidin complex 570 that includes an avidin protein bound to four biotin linking moieties.

[0180] In some embodiments, the shielding element 552 comprises an avidin protein that includes one or two non-functional binding sites engineered into the avidin protein. For example, in some embodiments, the shielding element 552 comprises a bivalent avidin complex 572 that includes an avidin protein bound to a biotin linking moiety on each of two subunits, where the avidin protein includes a non-functional ligand binding site 548 on each of two other subunits. As shown, in some embodiments, the bivalent avidin complex 572 includes a trans bivalent avidin protein, although a cis bivalent avidin protein may be used depending on the desired implementation. In some embodiments, the avidin protein is a trivalent avidin protein. In some embodiments, the trivalent avidin protein includes a non-functional ligand binding site 548 on one subunit and is bound to three biotin linking moieties or is bound to a biotin linking moiety and a bis-biotin linking moiety on one subunit.

[0181] In some embodiments, the shielding element 552 comprises a dendritic polymer 574. In some embodiments, the dendritic polymer 574 is a polyol or a dendrimer as described elsewhere herein. In some embodiments, the dendritic polymer 574 is a branched polyol or a branched dendrimer. In some embodiments, the dendritic polymer 574 comprises a monosaccharide-TEG, a disaccharide, an N-acetyl monosaccharide, a TEMPO-TEG, a Trolox-TEG, or a glycerol dendrimer. Examples of useful polyols according to the shield recognition molecules of the present application include polyether polyols and polyester polyols, such as polyethylene glycol, polypropylene glycol, and similar such polymers known in the art. In some embodiments, the dendritic polymer 574 is a polyol having the formula: -(CH 2 CH 2 In some embodiments, dendritic polymer 574 comprises a compound of the formula: -(CH2 CH 2 O) n-, where n is an integer from 1 to 100, inclusive.

[0182] In some embodiments, the shielding element 552 comprises a nucleic acid. In some embodiments, the nucleic acid is single stranded. In some embodiments, the label moiety 554 is attached directly or indirectly to one end (e.g., the 5' end or the 3' end) of the single stranded nucleic acid, and the recognition moiety 550 is attached directly or indirectly to the other end (e.g., the 3' end or the 5' end) of the single stranded nucleic acid. For example, a single stranded nucleic acid can include a label attached to the 5' end of the nucleic acid and an amino acid recognition molecule attached to the 3' end of the nucleic acid.

[0183] In some embodiments, the shielding element 552 comprises a double-stranded nucleic acid 576. As shown, in some embodiments, the double-stranded nucleic acid 576 is capable of forming a non-covalent linkage between the recognition moiety 550 and the label moiety 554 via strand hybridization. However, in some embodiments, the double-stranded nucleic acid 576 is capable of forming a covalent linkage between the recognition moiety 550 and the label moiety 554 via attachment to the same oligonucleotide strand. In some embodiments, the label moiety 554 is attached directly or indirectly to one end of the double-stranded nucleic acid, and the recognition moiety 550 is attached directly or indirectly to the other end of the double-stranded nucleic acid. For example, the double-stranded nucleic acid may comprise a label attached to the 5' end of one strand and an amino acid recognition molecule attached to the 5' end of the other strand.

[0184] In some embodiments, the shielding element 552 comprises nucleic acids that form one or more structural motifs that may be useful for increasing the steric bulk of the shield. Examples of nucleic acid structural motifs include, but are not limited to, stem-loops, three-way junctions (e.g., formed by two or more stem-loop motifs), four-way junctions (e.g., Holliday junctions), and bulge loops.

[0185] In some embodiments, the shielding element 552 comprises a nucleic acid that forms a stem-loop 578. A stem-loop or hairpin loop is an unpaired loop of nucleotides on an oligonucleotide strand that is formed when the oligonucleotide strand folds and base pairs with another section of the same strand. In some embodiments, the unpaired loop of the stem-loop 578 comprises 3-10 nucleotides. Thus, the stem-loop 578 can be formed by two regions of an oligonucleotide strand that have inverted complementary sequences that hybridize to form a stem, where the two regions are separated by 3-10 nucleotides that form an unpaired loop. In some embodiments, the stem of the stem-loop 578 can be designed to have one or more G / C nucleotides that can provide increased stability due to additional hydrogen bonding interactions that occur compared to A / T nucleotides. In some embodiments, the stem of the stem-loop 578 comprises a G / C nucleotide immediately adjacent to the unpaired loop sequence. In some embodiments, the stem of the stem-loop 578 comprises a G / C nucleotide within the first 2, 3, 4, or 5 nucleotides adjacent to the unpaired loop sequence. In some embodiments, the unpaired loop of stem-loop 578 comprises one or more attachment sites. In some embodiments, the attachment site occurs at an abasic site in the unpaired loop. In some embodiments, the attachment site occurs at a base of the unpaired loop.

[0186] In some embodiments, stem-loop 578 is formed by a double-stranded nucleic acid. As described herein, in some embodiments, double-stranded nucleic acid can form a non-covalent linking group through strand hybridization of a first and second oligonucleotide strand. However, in some embodiments, shielding element 552 includes a single-stranded nucleic acid that forms a stem-loop motif to provide a covalent linking group. In some embodiments, shielding element 552 includes a nucleic acid that forms two or more stem-loop motifs. For example, in some embodiments, the nucleic acid includes two stem-loop motifs. In some embodiments, the stem of one stem-loop motif is adjacent to the stem of the other such that the motifs together form a three-way junction. In some embodiments, shielding element 552 includes a nucleic acid that forms a four-way junction 578. In some embodiments, four-way junction 578 is formed through hybridization of two or more oligonucleotide strands (e.g., two, three, or four oligonucleotide strands).

[0187] In some embodiments, the shielding element 552 comprises one or more polymers selected from 560, 562, 564, 566, 568, 570, 572, 574, 576, 578, and 580 in Figure 5E. It should be appreciated that the connection portions and attachment sites shown in each of 560, 562, 564, 566, 568, 570, 572, 574, 576, 578, and 580 are shown for illustrative purposes and are not intended to depict preferred connection site or attachment site configurations.

[0188] In some embodiments, the present application provides a compound of formula (I): A-(Y) n -D (I) wherein A is an amino acid binding moiety comprising at least one amino acid recognition molecule, each Y is a polymer forming a covalent or non-covalent linking group, n is an integer from 1 to 10 inclusive, and D is a labeling moiety comprising at least one detectable label. In some embodiments, the present application provides a composition comprising a soluble amino acid recognition molecule of formula (I).

[0189] In some embodiments, A comprises a plurality of amino acid recognition molecules. In some embodiments, each amino acid recognition molecule of the plurality is attached to a different attachment site on Y. In some embodiments, at least two of the amino acid recognition molecules of the plurality are attached to a single attachment site on Y. In some embodiments, the amino acid recognition molecules are recognition proteins or nucleic acid aptamers, such as those described elsewhere herein.

[0190] In some embodiments, the detectable label is a luminescent label or a conductivity label. In some embodiments, the luminescent label comprises at least one fluorophore dye molecule. In some embodiments, D comprises 20 or less fluorophore dye molecules. In some embodiments, the ratio of the number of fluorophore dye molecules to the number of amino acid recognition molecules is 1:1 to 20:1. In some embodiments, the luminescent label comprises at least one FRET pair comprising a donor label and an acceptor label. In some embodiments, the ratio of the donor label to the acceptor label is 1:1, 2:1, 3:1, 4:1, or 5:1. In some embodiments, the ratio of the acceptor label to the donor label is 1:1, 2:1, 3:1, 4:1, or 5:1.

[0191] In some embodiments, D is a diameter of less than 200 Å. In some embodiments, -(Y) n - is at least 2 nm in length. In some embodiments, -(Y) n - is at least 5 nm in length. In some embodiments, -(Y) n- is at least 10 nm in length. In some embodiments, each Y is independently a biomolecule, a polyol, or a dendrimer. In some embodiments, the biomolecule is a nucleic acid, a polypeptide, or a polysaccharide.

[0192] In some embodiments, the amino acid recognition molecule has the formula: AY 1 -(Y) m -D or A-(Y) m -Y 1 -D (In the formula, Y 1 is a nucleic acid or a polypeptide, and m is an integer between 0 and 10 (inclusive). It is one of the following.

[0193] In some embodiments, the nucleic acid comprises a first oligonucleotide strand. In some embodiments, the nucleic acid comprises a second oligonucleotide strand hybridized to the first oligonucleotide strand. In some embodiments, the nucleic acid forms a covalent linkage through the first oligonucleotide strand. In some embodiments, the nucleic acid forms a non-covalent linkage through the hybridized first and second oligonucleotide strands.

[0194] In some embodiments, the polypeptide is a monovalent or multivalent protein. In some embodiments, the monovalent or multivalent protein forms at least one non-covalent linkage via a ligand moiety attached to a ligand binding site of the monovalent or multivalent protein. In some embodiments, A, Y, or D comprises a ligand moiety.

[0195] In some embodiments, the amino acid recognition molecule has the formula: A-(Y) m -Y 2 -D or AY 2 -(Y) m -D (In the formula, Y 2 is a polyol or a dendrimer, and m is an integer from 0 to 10 (inclusive). In some embodiments, the polyol or dendrimer comprises polyethylene glycol, tetraethylene glycol, poly(amidoamine), poly(propyleneimine), poly(propyleneamine), carbosilane, poly(L-lysine), or a combination of one or more thereof.

[0196] In some embodiments, the present application provides a compound of formula (II): AY 1 -D (II) where A is an amino acid binding moiety that includes at least one amino acid recognition molecule, and Y 1 is a nucleic acid or polypeptide, and D is a labeling moiety comprising at least one detectable label. In some embodiments, the amino acid recognition molecule is 1 When Y is a nucleic acid, the nucleic acid forms a covalent or non-covalent linking group. 1 is a polypeptide, the polypeptide is 50×10 -9 Dissociation constant (K D ) to form a non-covalent linking group characterized by

[0197] In some embodiments, Y 1 is a nucleic acid comprising a first oligonucleotide strand. In some embodiments, the nucleic acid comprises a second oligonucleotide strand hybridized to the first oligonucleotide strand. In some embodiments, A is attached to the first oligonucleotide strand and D is attached to the second oligonucleotide strand. In some embodiments, A is attached to a first attachment site on the first oligonucleotide strand and D is attached to a second attachment site on the first oligonucleotide strand. In some embodiments, each oligonucleotide strand of the nucleic acid comprises less than 150, less than 100, or less than 50 nucleotides.

[0198] In some embodiments, Y 1is a monovalent or multivalent protein. In some embodiments, the monovalent or multivalent protein forms at least one non-covalent linkage via a ligand moiety attached to a ligand binding site of the monovalent or multivalent protein. In some embodiments, at least one of A and D comprises a ligand moiety. In some embodiments, the polypeptide is an avidin protein (e.g., avidin, streptavidin, traptavidin, tamavidin, bladdavidin, xenavidin, or homologs or variants thereof). In some embodiments, the ligand moiety is a biotin moiety.

[0199] In some embodiments, the amino acid recognition molecule has the formula: AY 1 -(Y) n -D or A-(Y) n -Y 1 -D wherein each Y is a polymer that forms a covalent or non-covalent linking group, and n is an integer from 1 to 10, inclusive. In some embodiments, each Y is independently a biomolecule, a polyol, or a dendrimer.

[0200] In another aspect, the present application provides an amino acid recognition molecule comprising a nucleic acid, at least one amino acid recognition molecule attached to a first attachment site on the nucleic acid, and at least one detectable label attached to a second attachment site on the nucleic acid. In some embodiments, the nucleic acid forms a covalent or non-covalent linking group between the at least one amino acid recognition molecule and the at least one detectable label.

[0201] In some embodiments, the nucleic acid is a double-stranded nucleic acid comprising a first oligonucleotide strand hybridized to a second oligonucleotide strand. In some embodiments, the first attachment site is on the first oligonucleotide strand and the second attachment site is on the second oligonucleotide strand. In some embodiments, the at least one amino acid recognition molecule is attached to the first attachment site via a protein that forms a covalent or non-covalent linking group between the at least one amino acid recognition molecule and the nucleic acid. In some embodiments, the at least one detectable label is attached to the second attachment site via a protein that forms a covalent or non-covalent linking group between the at least one detectable label and the nucleic acid. In some embodiments, the first and second attachment sites are separated by 5-100 nucleotide bases or nucleotide base pairs on the nucleic acid.

[0202] In yet another aspect, the present application provides an amino acid recognition molecule comprising a multivalent protein comprising at least two ligand binding sites, at least one amino acid recognition molecule attached to the protein via a first ligand moiety bound to a first ligand binding site on the protein, and at least one detectable label attached to the protein via a second ligand moiety bound to a second ligand binding site on the protein.

[0203] In some embodiments, the multivalent protein is an avidin protein that comprises four ligand binding sites. In some embodiments, the ligand binding site is a biotin binding site, and the ligand moiety is a biotin moiety. In some embodiments, at least one of the biotin moieties is a bis-biotin moiety, and the bis-biotin moiety is bound to two biotin binding sites on the avidin protein. In some embodiments, at least one amino acid recognition molecule is attached to the protein via a nucleic acid that comprises a first ligand moiety. In some embodiments, at least one detectable label is attached to the protein via a nucleic acid that comprises a second ligand moiety.

[0204] As described elsewhere herein, the shield recognition molecules of the present application may be used in the polypeptide sequencing methods of the present application or any method known in the art. For example, in some embodiments, the shield recognition molecules provided herein may be used in Edman-type degradation reactions as provided herein or conventionally known in the art, which may involve repeated cycling of multiple reaction mixtures in a polypeptide sequencing reaction. In some embodiments, the shield recognition molecules provided herein may be used in kinetic sequencing reactions of the present application, which involve amino acid recognition and degradation in a single reaction mixture.

[0205] Sequencing by degradation of labeled polypeptides In some aspects, the present application provides a method for sequencing a polypeptide by identifying a unique combination of amino acids that correspond to a known polypeptide sequence. For example, FIG. 6 shows a method for sequencing by selectively detecting labeled amino acids of a labeled polypeptide 600. In some embodiments, the labeled polypeptide 600 comprises amino acids that are selectively modified such that different amino acid types contain different luminescent labels. As used herein, unless otherwise specified, a labeled polypeptide refers to a polypeptide that comprises one or more selectively labeled amino acid side chains. Details regarding the method of selective labeling and the preparation and analysis of labeled polypeptides are known in the art (see, for example, Swaminathan et al., PLoS Comput Biol., 2015, Vol. 11, No. 2, p. e1004080).

[0206] As shown, in some embodiments, the labeled polypeptide 600 is immobilized and exposed to an excitation source. Aggregate luminescence from the labeled polypeptide 600 is detected, and in some embodiments, exposure to luminescence over time results in a loss of detection signal due to luminescence label degradation (e.g., degradation due to photobleaching). In some embodiments, the labeled polypeptide 600 includes a unique combination of selectively labeled amino acids that generate an initial detection signal. As generally exemplified, luminescence label degradation over time results in a corresponding decrease in detection signal of the photobleached labeled polypeptide 602. In some embodiments, the signal can be deconvoluted by analysis of one or more luminescence properties (e.g., signal deconvolution by luminescence lifetime analysis). In some embodiments, the unique combination of selectively labeled amino acids of the labeled polypeptide 600 has been pre-calculated and empirically verified, for example, on a computer based on known polypeptide sequences of the proteome. In some embodiments, the combination of detected amino acid labels is compared to a database of known sequences of the proteome of an organism to identify a particular polypeptide in the database that corresponds to the labeled polypeptide 600.

[0207] In some embodiments, the approach illustrated in FIG. 6 can be modified by determining the optimal sample concentration to perform a sequencing reaction that maximizes sampling in a massively parallel analysis. In some embodiments, the concentration is selected so that a desired portion (e.g., 30%) of the sample wells of the array are occupied at any given time. Without wishing to be bound by theory, it is believed that as a polypeptide is bleached over a period of time, the same wells are continuously available for further analysis. Through diffusion, approximately 30% of the sample wells of the array are available for analysis every 3 minutes. As an illustrative example, a 1 million sample well chip can sample 6,000,000 polypeptides per hour or 24,000,000 polypeptides over 4 hours.

[0208] In some aspects, the present application provides methods for sequencing a polypeptide by detecting the emission of light from a labeled polypeptide that is subjected to repeated cycles of terminal amino acid modification and cleavage. For example, FIG. 7 illustrates a method for sequencing a labeled polypeptide by Edman degradation according to the present application. In some embodiments, the method proceeds generally as described herein for other sequencing methods by Edman degradation. For example, in some embodiments, steps (1) and (2) shown in FIG. 7 may be performed as described elsewhere herein for terminal amino acid modification and terminal amino acid cleavage, respectively, in an Edman degradation reaction.

[0209] As shown in the example depicted in FIG. 7, in some embodiments, the method includes (1) modifying a terminal amino acid of the labeled polypeptide. As described elsewhere herein, in some embodiments, modifying includes contacting the terminal amino acid with an isothiocyanate (e.g., PITC) to form an isothiocyanate-modified terminal amino acid. In some embodiments, the isothiocyanate modification 710 converts the terminal amino acid into a form that is more susceptible to removal by a cleavage reagent (e.g., a chemical or enzymatic cleavage reagent described herein). Thus, in some embodiments, the method includes (2) removing the modified terminal amino acid using chemical or enzymatic means as detailed elsewhere herein for Edman degradation.

[0210] In some embodiments, the method includes repeating steps (1)-(2) for multiple cycles during which light emission from the labeled polypeptide is detected and cleavage events corresponding to removal of the labeled amino acid from the terminus can be detected as a decrease in the detection signal. In some embodiments, if the signal does not change following step (2) shown in FIG. 7, an amino acid of unknown type is identified. Thus, in some embodiments, partial sequence information can be determined by evaluating the signal detected following step (2) at each successive round and assigning an amino acid type with an identity determined based on a change in the detection signal, or identifying the amino acid type as unknown based on no change in the detection signal.

[0211] In some aspects, methods of sequencing polypeptides according to the present application include sequencing by processive enzymatic cleavage of a labeled polypeptide, as generally illustrated in Figures 8A-8C. As shown, in some embodiments, the labeled polypeptide is subjected to degradation with a modified processive exopeptidase that sequentially cleaves terminal amino acids from one end to the other. Exopeptidases are described in detail elsewhere herein. Figure 8A depicts an example in which a labeled polypeptide 800 is subjected to degradation by an immobilized processive exopeptidase 810. Figure 8B depicts an example in which an immobilized labeled polypeptide 820 is subjected to degradation by a processive exopeptidase 830.

[0212] FIG. 8C illustrates a schematic example of a real-time sequencing process carried out according to the method depicted in FIG. 8B. As shown, panels (I)-(IV) show the progression of labeled polypeptide degradation, with the corresponding signal traces shown below each panel over time. As shown, each cleavage event corresponding to a labeled amino acid causes a concomitant decrease in signal. In some embodiments, since the processivity rate of the processive exopeptidase 830 is known, the timing of the decrease in detected signal can be used to calculate the number of unlabeled amino acids between each detection event. For example, if a 40 amino acid polypeptide is cleaved such that an amino acid is removed every second, a labeled polypeptide with 3 signals will initially show all 3 signals (panel (I)), then 2 signals (panel (II)), then 1 signal (panel (III)), and finally no signal. Thus, the order of the labeled amino acids can be determined. These methods can therefore be used, for example, in proteomic analysis based on polypeptide fragment sequencing to determine partial sequence information.

[0213] In some embodiments, single molecule protein sequencing can be accomplished using an ATP-based Förster resonance energy transfer (FRET) scheme (e.g., with one or more labeled cofactors), for example, as illustrated in Figure 9. In some embodiments, sequencing by cofactor-based FRET can be performed using an immobilized ATP-dependent protease, donor-labeled ATP, and acceptor-labeled amino acids of a polypeptide substrate. In some embodiments, the amino acid can be labeled with an acceptor, and one or more cofactors can be labeled with a donor.

[0214] For example, in some embodiments, extracted proteins are denatured and cysteines and lysines are labeled with fluorescent dyes. In some embodiments, engineered protein translocases (e.g., bacterial ClpX) are used to bind individual substrate proteins, unfold them, and translocate them through the nanochannel. In some embodiments, the translocase is labeled with a donor dye, and FRET occurs between the donor on the translocase and two or more distinguishable acceptor dyes on the substrate as the substrate passes through the nanochannel. The order of the labeled amino acids can then be determined from the FRET signal. In some embodiments, one or more of the following non-limiting labeled ATP analogs shown in Table 5 can be used:

[0215] [Table 5-1]

[0216] [Table 5-2]

[0217] [Table 5-3]

[0218] Preparation of samples for sequencing The polypeptide sample can be modified prior to sequencing. In some embodiments, the N-terminal or C-terminal amino acid of the polypeptide can be modified. FIG. 10A illustrates a non-limiting example of terminal modification for preparing terminally modified polypeptides from a protein sample. In step (1), the protein sample 1000 is fragmented to generate polypeptide fragments 1002. The polypeptide can be fragmented by cleaving (e.g., chemically) and / or digesting (e.g., enzymatically using a peptidase such as trypsin) the polypeptide of interest. Fragmentation can be performed before or after labeling. In some embodiments, fragmentation is performed after labeling of the whole protein. One or more amino acids can be labeled before or after cleavage to generate a labeled polypeptide. In some embodiments, the polypeptide is size selected after chemical or enzymatic fragmentation. In some embodiments, smaller polypeptides (e.g., <2 kDa) are removed and larger polypeptides are retained for sequence analysis. Size selection can be achieved using techniques such as gel filtration, SEC, dialysis, PAGE gel extraction, microfluidic tension flow, or any other suitable technique. In step (2), the N-terminus or C-terminus of the polypeptide fragment 1002 is modified to generate a terminally modified polypeptide 1004. In some embodiments, the modification comprises adding an anchoring moiety. In some embodiments, the modification comprises adding a coupling moiety.

[0219] Thus, provided herein are methods for modifying the termini of proteins and polypeptides with moieties that allow for immobilization to a surface (e.g., the surface of a sample well on a chip used for protein analysis). In some embodiments, such methods involve modifying the termini of a labeled polypeptide to be analyzed in accordance with the present application. In yet other embodiments, such methods involve modifying the termini of a protein or an enzyme that degrades or translocates a polypeptide substrate in accordance with the present application.

[0220] In some embodiments, the carboxy terminus of a protein or polypeptide is modified by a method comprising: (i) blocking free carboxylic acid groups of the protein or polypeptide, (ii) denaturing the protein or polypeptide (e.g., by thermal and / or chemical means), (iii) blocking free thiol groups of the protein or polypeptide, (iv) digesting the protein or polypeptide to generate at least one polypeptide fragment comprising a free C-terminal carboxylic acid group, and (v) conjugating a functional moiety to the free C-terminal carboxylic acid group (e.g., chemically). In some embodiments, the method further comprises dialyzing a sample comprising the protein or polypeptide after (i) and prior to (ii).

[0221] In some embodiments, the carboxy terminus of a protein or polypeptide is modified by a method comprising: (i) denaturing the protein or polypeptide (e.g., by thermal and / or chemical means), (ii) blocking free thiol groups of the protein or polypeptide, (iii) digesting the protein or polypeptide to generate at least one polypeptide fragment comprising a free C-terminal carboxylic acid group, (iv) blocking the free C-terminal carboxylic acid group to generate at least one polypeptide fragment comprising a blocked C-terminal carboxylic acid group, and (v) conjugating a functional moiety to the blocked C-terminal carboxylic acid group (e.g., enzymatically). In some embodiments, the method further comprises dialyzing the sample comprising the protein or polypeptide after (iv) and prior to (v).

[0222] In some embodiments, blocking a free carboxylic acid group refers to a chemical modification of this group that changes its chemical reactivity compared to the unmodified carboxylic acid. Suitable carboxylic acid blocking methods are known in the art and should modify the side chain carboxylic acid group so that it is chemically different from the carboxy-terminal carboxylic acid group of the polypeptide to be functionalized. In some embodiments, blocking a free carboxylic acid group includes esterifying or amidating the free carboxylic acid group of the polypeptide. In some embodiments, blocking a free carboxylic acid group includes methyl esterifying the free carboxylic acid group of the polypeptide, for example, by reacting the polypeptide with methanolic HCl. Additional examples of reagents and techniques useful for blocking free carboxylic acid groups include, but are not limited to, 4-sulfo-2,3,5,6-tetrafluorophenol (STP) and / or carbodiimides such as N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDAC), uronium reagents, diazomethane, alcohols and acids for Fischer esterification, the use of N-hydroxylsuccinimide (NHS) to form NHS esters (potentially as an intermediate to subsequent ester or amine formation), or reaction with carbonyldiimidazole (CDI), or the formation of mixed anhydrides, or potentially any other method of modifying or blocking a carboxylic acid via the formation of either an ester or amide.

[0223] In some embodiments, blocking a free thiol group refers to a chemical modification of this group that changes its chemical reactivity compared to the unmodified thiol. In some embodiments, blocking a free thiol group includes reducing and alkylating the free thiol group of a protein or polypeptide. In some embodiments, the reduction and alkylation is performed by contacting the polypeptide with dithiothreitol (DTT) and one or both of iodoacetamide and iodoacetic acid. Examples of additional and alternative cysteine ​​reducing reagents that can be used are well known and include, but are not limited to, 2-mercaptoethanol, tris(2-carboxyethyl)phosphine hydrochloride (TCEP), tributylphosphine, dithiobutylamine (DTBA), or any reagent capable of reducing a thiol group. Examples of additional and alternative cysteine ​​blocking (e.g., cysteine ​​alkylating) reagents that can be used are well known and include, but are not limited to, acrylamide, 4-vinylpyridine, N-ethylmaleimide (NEM), N-ε-maleimidocaproic acid (EMCA), or any reagent that modifies cysteines to prevent disulfide bond formation.

[0224] In some embodiments, the digestion comprises enzymatic digestion. In some embodiments, the digestion is performed by contacting the protein or polypeptide with an endopeptidase (e.g., trypsin) under digestive conditions. In some embodiments, the digestion comprises chemical digestion. Examples of reagents suitable for chemical and enzymatic digestion are known in the art and include, but are not limited to, trypsin, chemotrypsin, Lys-C, Arg-C, Asp-N, Lys-N, BNPS-skatole, CNBr, caspase, formic acid, glutamyl endopeptidase, hydroxylamine, iodosobenzoic acid, neutrophil elastase, pepsin, proline-endopeptidase, proteinase K, staphylococcal peptidase I, thermolysin, and thrombin.

[0225] In some embodiments, the functional moiety comprises a biotin molecule. In some embodiments, the functional moiety comprises a reactive chemical moiety, such as alkynyl. In some embodiments, conjugating the functional moiety comprises biotinylation of the carboxy-terminal carboxymethyl ester group with carboxypeptidase Y, as known in the art.

[0226] In some embodiments, a solubilizing moiety is added to a polypeptide. Figure 10B illustrates a non-limiting example of a solubilizing moiety that is added to the terminal amino acid of a polypeptide, for example, using a process of conjugating a solubilizing linker to the polypeptide.

[0227] In some embodiments, a terminally modified polypeptide 1010 comprising a linker-conjugating moiety 1012 is conjugated to a solubilizing linker 1020 comprising a polypeptide-conjugating moiety 1022. In some embodiments, the solubilizing linker comprises a solubilizing polymer such as a biomolecule (e.g., shown as a speckled shape). In some embodiments, the resulting linker-conjugated polypeptide 1030 comprising a linkage 1032 formed between 1012 and 1022 further comprises a surface-conjugating moiety 1034. Thus, in some embodiments, the methods and compositions provided herein are useful for modifying the termini of a polypeptide with moieties that increase its solubility. In some embodiments, solubilizing moieties are useful for relatively insoluble small polypeptides resulting from fragmentation (e.g., enzymatic fragmentation, such as with trypsin). For example, in some embodiments, short polypeptides in a polypeptide pool can be solubilized by conjugating a polymer (e.g., short oligos, sugars, or other charged polymers) to the polypeptide.

[0228] In some embodiments, one or more surfaces of the sample well (e.g., the sidewalls of the sample well) can be modified. A non-limiting example of passivation and / or anti-fouling of the sample well sidewalls is shown in FIG. 10C, which illustrates an example schematic of a sample well with a modified surface that can be used to facilitate single molecule immobilization to the bottom surface. In some embodiments, 1040 is a SiO 2 In some embodiments, 1042 is a polypeptide conjugating moiety (e.g., TCO, tetrazine, N3, alkyne, aldehyde, NCO, NHS, thiol, alkene, DBCO, BCN, TPP, biotin, or other suitable conjugating moiety). In some embodiments, 1050 is TiO 2 Or Al 2 O 3 In some embodiments, 1052 is a hydrophobic C 4~18 molecules, polytetrafluoroethylene compounds (e.g., (CF 2 ) 4~12 ), polyols, such as polyethylene glycols (e.g., PEG 3~100 ), polypropylene glycol, polyoxyethylene glycol, or combinations or variations thereof, or zwitterions such as sulfobetaine. In some embodiments, 1060 is Si. In some embodiments, 1070 is Al. In some embodiments, 1080 is TiN.

[0229] Luminous indicator As used herein, a luminescent label is a molecule that can absorb one or more photons and subsequently emit one or more photons after one or more time durations. In some embodiments, the term is used synonymously with "label" or "luminescent molecule" depending on the context. A luminescent label according to certain embodiments described herein can refer to a luminescent label of a labeled affinity reagent, a luminescent label of a labeled peptidase (e.g., a labeled exopeptidase, a labeled non-specific exopeptidase), a luminescent label of a labeled peptide, a luminescent label of a labeled cofactor, or other labeling composition described herein. In some embodiments, a luminescent label according to the present application refers to a labeled amino acid of a labeled polypeptide that includes one or more labeled amino acids.

[0230] In some embodiments, the luminescent label may include a first and a second chromophore. In some embodiments, the excited state of the first chromophore may be relaxed via energy transfer to the second chromophore. In some embodiments, the energy transfer is Förster resonance energy transfer (FRET). Such FRET pairs may be useful to provide luminescent labels with properties that make label-by-label discrimination between multiple luminescent labels in a mixture easier, for example, as illustrated and described herein for labeled aptamer 106 of FIG. 1C. In yet other embodiments, the FRET pair includes a first chromophore of the first luminescent label and a second chromophore of the second luminescent label, for example, as illustrated and described herein for sequencing labeled peptides with a labeling cofactor (see, for example, FIG. 9). In certain embodiments, the FRET pair may absorb excitation energy in a first spectral region and emit emission in a second spectral region.

[0231] In some embodiments, luminescent label refers to a fluorophore or dye. Typically, luminescent labels include aromatic or heteroaromatic compounds and may be pyrene, anthracene, naphthalene, naphthylamine, acridine, stilbene, indole, benzoindole, oxazole, carbazole, thiazole, benzothiazole, benzoxazole, phenanthridine, phenoxazine, porphyrin, quinoline, ethidium, benzamide, cyanine, carbocyanine, salicylate, anthranilate, coumarin, fluorescein, rhodamine, xanthene, or other similar compounds.

[0232] In some embodiments, the luminescent label is one of the following: 5 / 6-carboxyrhodamine 6G, 5-carboxyrhodamine 6G, 6-carboxyrhodamine 6G, 6-TAMRA, Aberior® STAR 440SXP, Aberior® STAR 470SXP, Aberior® STAR 488, Aberior® STAR (ST AR) 512, Aberior® STAR 520SXP, Aberior® STAR 580, Aberior® STAR 600, Aberior® STAR 635, Aberior® STAR 635P, Aberior® STAR Red (STAR RED), Alexa Fluor® 350, Alexa Fluor® 405, Alexa Fluor® 430, Alexa Fluor® 480, Alexa Fluor® 488, Alexa Fluor® 514, Alexa Fluor® 532, Alexa Fluor® 546, Alexa Fluor® 555, Alexa Fluor® 568, Alexa Fluor® 594, Alexa Fluor® 610X, Alexa Fluor® 63 ... Alexa Fluor® 647, Alexa Fluor® 660, Alexa Fluor® 680, Alexa Fluor® 700, Alexa Fluor® 750,Alexa Fluor® 790, AMCA, ATTO 390, ATTO 425, ATTO 465, ATTO 488, ATTO 495, ATTO 514, ATTO 520, ATTO 532, ATTO 542, ATTO 550, ATTO 565, ATTO 590, ATTO 610, ATTO 620, ATTO 633, ATTO 647, ATTO 647N ... ATTO 655, ATTO 665, ATTO 680, ATTO 700, ATTO 725, ATTO 740, ATTO Oxa12, ATTO Rho101, ATTO Rho11, ATTO Rho12, ATTO Rho13, ATTO Rho14, ATTO Rho3B, ATTO Rho6G, ATTO Thio12, BD Horizon™ V450, BodiP( BODIPY® 493 / 501, BODIPY® 530 / 550, BODIPY® 558 / 568, BODIPY® 564 / 570, BODIPY® 576 / 589, BODIPY® 581 / 591, BODIPY® 630 / 650, BODIPY® 650 / 665, BODIPY® FL, BODIPY ) FL-X, BODIPY R6G, BODIPY TMR, BODIPY TR, CAL Fluor Gold 540, CAL Fluor Green 510, CAL Fluor Orange 560, CAL Fluor Red 590, CAL Fluor Red 610, CAL Fluor Red 615,CAL Fluor® Red 635, Cascade® Blue, CF™ 350, CF™ 405M, CF™ 405S, CF™ 488A, CF™ 514, CF™ 532, CF™ 543, CF™ 546, CF™ 555, CF™ 568, CF™ 594, CF™ 620R, CF™ 633, CF™ 633-V1, CF™ 640R, CF™ 640R-V1, CF™ 640R-V2, CF™ 660C, CF™ 660R, CF™ 680, CF™ 680R, CF™ 680R-V1, CF™ 750, CF™ 770, CF™ 790, Chromeo™ 642, Chromis 425N, Chromis 500N, Chromis 515N, Chromis 530N, Chromis 550A, Chromis 550C, Chromis 550Z, Chromis 560N, Chromis (Chr chromis 570N, Chromis 577N, Chromis 600N, Chromis 630N, Chromis 645A, Chromis 645C, Chromis 645Z, Chromis 678A, Chromis 678C, Chromis 678Z, Chromis 770A, Chromis 770C, Chromis 800A, Chromis 800C, Chromis (Chromis) 830A, Chromis 830C, Cy® 3, Cy® 3.5, Cy® 3B, Cy® 5, Cy® 5.5, Cy® 7, DyLight® 350, DyLight® 405, DyLight® 415-Co1, DyLight® 425Q, DyLight® 485-LS, DyLight® 488,DyLight® 504Q, DyLight® 510-LS, DyLight® 515-LS, DyLight® 521-LS, DyLight® 530-R2, DyLight® 543Q, DyLight® 550, DyLight® 554-R0, DyLight® 554-R1, DyLight DyLight 590-R2, DyLight 594, DyLight 610-B1, DyLight 615-B2, DyLight 633, DyLight 633-B1, DyLight 633-B2, DyLight 650, DyLight 655-B1, DyLight 655-B2, DyLight DyLight (registered trademark) 655-B3, DyLight (registered trademark) 655-B4, DyLight (registered trademark) 662Q, DyLight (registered trademark) 675-B1, DyLight (registered trademark) 675-B2, DyLight (registered trademark) 675-B3, DyLight (registered trademark) 675-B4, DyLight (registered trademark) 679-C5, DyLight (registered trademark) 680, DyLight (registered trademark) DyLight (registered trademark) 683Q, DyLight (registered trademark) 690-B1, DyLight (registered trademark) 690-B2, DyLight (registered trademark) 696Q, DyLight (registered trademark) 700-B1, DyLight (registered trademark) 700-B1, DyLight (registered trademark) 730-B1, DyLight (registered trademark) 730-B2, DyLight (registered trademark) 730-B3, DyLight (registered trademark) 730-B4,DyLight® 747, DyLight® 747-B1, DyLight® 747-B2, DyLight® 747-B3, DyLight® 747-B4, DyLight® 755, DyLight® 766Q, DyLight® 775-B2, DyLight® 775-B3, DyLight® ht) 775-B4, DyLight 780-B1, DyLight 780-B2, DyLight 780-B3, DyLight 800, DyLight 830-B2, Dyomics-350, Dyomics-350XL, Dyomics-360XL, Dyomics-370XL, Dyomics Dyomics-375XL, Dyomics-380XL, Dyomics-390XL, Dyomics-405, Dyomics-415, Dyomics-430, Dyomics-431, Dyomics-478, Dyomics-480XL, Dyomics-481XL, Dyomics-485XL, Dyomics-490, Dyomics (Dyomics)-495, Dyomics-505, Dyomics-510XL, Dyomics-511XL, Dyomics-520XL, Dyomics-521XL, Dyomics-530, Dyomics-547, Dyomics-547P1, Dyomics-548, Dyomics-549, Dyomics-549P1,Dyomics-550, Dyomics-554, Dyomics-555, Dyomics-556, Dyomics-560, Dyomics-590, Dyomics-591, Dyomics-594, Dyomics(Dy, Dyomics-601XL, Dyomics-605, Dyomics-610, Dyomics-615, Dyomics-630, Dyomics-631, Dyomics-632, Dyomics-633, Dyomics-634, Dyomics-635, Dyomics-636, Dyomics-647, Dyomics cs)-647P1, Dyomics-648, Dyomics-648P1, Dyomics-649, Dyomics-649P1, Dyomics-650, Dyomics-651, Dyomics-652, Dyomics-654, Dyomics-675, Dyomics-676, Dyomics-677, Dyomics cs)-678, Dyomics-679P1, Dyomics-680, Dyomics-681, Dyomics-682, Dyomics-700, Dyomics-701, Dyomics-703, Dyomics-704, Dyomics-730, Dyomics-731, Dyomics-732, Dyomics- 734, Dyomics-749, Dyomics-749P1, Dyomics-750, Dyomics-751, Dyomics-752, Dyomics-754, Dyomics-776, Dyomics-777, Dyomics-778, Dyomics-780, Dyomics-781, Dyomics-782,Dyomics-800, Dyomics-831, eFluor® 450, Eosin, FITC, Fluorescein, HiLyte™ Fluor 405, HiLyte™ Fluor 488, HiLyte™ Fluor 532, HiLyte™ Fluor 555, HiLyte™ Fluor 594, HiLyte™ Fluor 647, HiLyte™ Fluor 680 ...32, HiLyte™ Fluor 532, HiLyte™ Fluor 532, HiLyte™ Fluor 532, HiLy HiLyte™ Fluor 750, IRDye® 680LT, IRDye® 750, IRDye® 800CW, JOE, LightCycler® 640R, LightCycler® Red 610, LightCycler® Red 640, LightCycler® Red 670, LightCycler® Red 705, Lissamine Rhodamine B, Naphthofluorescein, Oregon Green Green® 488, Oregon Green® 514, Pacific Blue™, Pacific Green™, Pacific Orange™, PET, PF350, PF405, PF415, PF488, PF505, PF532, PF546, PF555P, PF568, PF594, PF610, PF633P, PF647P, Quasar® 570, Quasar® 670, Quasar® 705, Rhodamine 123, Rhodamine 6G, Rhodamine B, Rhodamine Green, Rhodamine Green-X, Rhodamine Red, ROX, Seta™ 375, Seta™ 470,Seta(TM) 555, Seta(TM) 632, Seta(TM) 633, Seta(TM) 650, Seta(TM) 660, Seta(TM) 670, Seta(TM) 680, Seta(TM) 700, Seta(TM) 750, Seta(TM) 780, Seta(TM) APC-780, Seta(TM) PerCP-680, Seta(TM) and / or a dye selected from one or more of Seta™ R-PE-670, Seta™ 646, SeTau 380, SeTau 425, SeTau 647, SeTau 405, Square 635, Square 650, Square 660, Square 672, Square 680, Sulforhodamine 101, TAMRA, TET, Texas Red®, TMR, TRITC, Yakima Yellow™, Zenon®, Zy3, Zy5, Zy5.5, and Zy7.

[0233] Illumination In some aspects, the present application relates to polypeptide sequencing and / or identification based on one or more luminescent properties of the luminescent label. In some embodiments, the luminescent label is identified based on luminescence lifetime, luminescence intensity, brightness, absorption spectrum, emission spectrum, luminescence quantum yield, or a combination of two or more thereof. In some embodiments, multiple types of luminescent labels are distinguishable from each other based on different luminescence lifetime, luminescence intensity, brightness, absorption spectrum, emission spectrum, luminescence quantum yield, or a combination of two or more thereof. Identifying can mean assigning the exact identity and / or amount of one type of amino acid (e.g., a single type or a subset of types) associated with the luminescent label, and can also mean assigning the amino acid position in the polypeptide relative to other types of amino acids.

[0234] In some embodiments, the luminescence is detected by exposing the luminescent label to a series of individual light pulses and evaluating the timing or other properties of each photon emitted from the label. In some embodiments, the information of the multiple photons sequentially emitted from the label is aggregated and evaluated to identify the label and thereby the type of related amino acid. In some embodiments, the luminescence lifetime of the label is determined from the multiple photons sequentially emitted from the label, and the luminescence lifetime can be used to identify the label. In some embodiments, the luminescence intensity of the label is determined from the multiple photons sequentially emitted from the label, and the luminescence intensity can be used to identify the label. In some embodiments, the luminescence lifetime and luminescence intensity of the label are determined from the multiple photons sequentially emitted from the label, and the luminescence lifetime and luminescence intensity can be used to identify the label.

[0235] In some aspects of the present application, a single polypeptide molecule is exposed to multiple individual light pulses, and the sequence of emitted photons is detected and analyzed. In some embodiments, the sequence of emitted photons provides information about a single polypeptide molecule present that does not change in the reaction sample over the time of the experiment. However, in some embodiments, the sequence of emitted photons provides information about a sequence of different molecules present in the reaction sample at different times (e.g., as a reaction or process progresses). By way of example and not limitation, such information may be used to sequence and / or identify polypeptides that have been subjected to chemical or enzymatic degradation in accordance with the present application.

[0236] In certain embodiments, a luminescent label absorbs a photon and emits a photon after a duration. In some embodiments, the luminescent lifetime of a label can be determined or estimated by measuring the duration. In some embodiments, the luminescent lifetime of a label can be determined or estimated by measuring multiple durations for multiple pulse and emission events. In some embodiments, the luminescent lifetime of a label can be distinguished between the luminescent lifetimes of multiple types of labels by measuring the duration. In some embodiments, the luminescent lifetime of a label can be distinguished between the luminescent lifetimes of multiple types of labels by measuring multiple durations for multiple pulse and emission events. In certain embodiments, a label is identified or distinguished between multiple types of labels by determining or estimating the luminescent lifetime of the label. In certain embodiments, a label is identified or distinguished between multiple types of labels by distinguishing the luminescent lifetime of a label between multiple luminescent lifetimes of multiple types of labels.

[0237] Determining the luminescence lifetime of a luminescent label can be performed using any suitable method (e.g., by measuring the lifetime using a suitable technique or by determining the time-dependent characteristics of the emission). In some embodiments, determining the luminescence lifetime of one label comprises determining the lifetime relative to another label. In some embodiments, determining the luminescence lifetime of a label comprises determining the lifetime relative to a reference. In some embodiments, determining the luminescence lifetime of a label comprises measuring a lifetime (e.g., a fluorescence lifetime). In some embodiments, determining the luminescence lifetime of a label comprises determining one or more time characteristics indicative of the lifetime. In some embodiments, the luminescence lifetime of a label can be determined based on a distribution of multiple emission events (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more emission events) occurring over one or more time-gated windows relative to an excitation pulse. For example, the luminescence lifetime of a label can be distinguished from multiple labels having different luminescence lifetimes based on the distribution of photon arrival times measured relative to an excitation pulse.

[0238] It should be appreciated that the luminescence lifetime of a luminescent label is indicative of the timing of photons emitted after the label reaches an excited state, and labels can be distinguished by information indicative of the timing of the photons. Some embodiments can include distinguishing a label from a plurality of labels based on the luminescence lifetime of the label by measuring the time associated with a photon emitted by the label. The distribution of times can provide an indication of the luminescence lifetime that can be determined from the distribution. In some embodiments, a label can be distinguished from a plurality of labels based on the distribution of times, for example, by comparing the distribution of times to a reference distribution corresponding to a known label. In some embodiments, a luminescence lifetime value has been determined from the distribution of times.

[0239] As used herein, in some embodiments, luminescence intensity refers to the number of emission photons per unit time emitted by a luminescent label that has been excited by the delivery of a pulse of excitation energy. In some embodiments, luminescence intensity refers to the number of detected emission photons per unit time emitted by a label that has been excited by the delivery of a pulse of excitation energy and detected by a particular sensor or set of sensors.

[0240] As used herein, in some embodiments, brightness refers to a parameter that reports on the average luminescence intensity per luminescent label. Thus, in some embodiments, "luminescence intensity" can be used to generally refer to the brightness of a composition that includes one or more labels. In some embodiments, the brightness of a label is equal to the product of its quantum yield and extinction coefficient.

[0241] As used herein, in some embodiments, luminescence quantum yield refers to the fraction of excitation events at a given wavelength or within a given spectral region that result in an emission event, and is typically less than 1. In some embodiments, the luminescence quantum yield of the luminescent labels described herein is 0 to about 0.001, about 0.001 to about 0.01, about 0.01 to about 0.1, about 0.1 to about 0.5, about 0.5 to 0.9, or about 0.9 to 1. In some embodiments, the labels are identified by identifying or estimating the luminescence quantum yield.

[0242] As used herein, in some embodiments, the excitation energy is a light pulse from a light source. In some embodiments, the excitation energy is in the visible spectral range. In some embodiments, the excitation energy is in the ultraviolet spectral range. In some embodiments, the excitation energy is in the infrared spectral range. In some embodiments, the excitation energy is at or near the absorption maximum of the luminescent label where multiple emission photons are detected. In certain embodiments, the excitation energy is about 500 nm to about 700 nm (e.g., about 500 nm to about 600 nm, about 600 nm to about 700 nm, about 500 nm to about 550 nm, about 550 nm to about 600 nm, about 600 nm to about 650 nm, or about 650 nm to about 700 nm). In certain embodiments, the excitation energy can be monochromatic or confined to a spectral range. In some embodiments, the spectral range has a range of about 0.1 nm to about 1 nm, about 1 nm to about 2 nm, or about 2 nm to about 5 nm. In some embodiments, the spectral region has a range from about 5 nm to about 10 nm, from about 10 nm to about 50 nm, or from about 50 nm to about 100 nm.

[0243] Sequencing Aspects of the present application relate to sequencing of biological polymers such as polypeptides and proteins. As used herein, the terms "sequencing", "sequencing", "determining a sequence" and the like, with respect to a polypeptide or protein, include determining partial sequence information of the polypeptide or protein, as well as the entire sequence information. That is, the terms include sequence comparison, fingerprinting, probabilistic fingerprinting, and the like, with respect to the target molecule, as well as the unambiguous identification and ordering of each amino acid of the target molecule within a region of interest. In some embodiments, the terms include identifying a single amino acid of a polypeptide. In still other embodiments, more than one amino acid of a polypeptide is identified. As used herein, in some embodiments, the terms "identifying", "determining an identity" and the like, with respect to an amino acid, include determining the unambiguous identity of the amino acid, as well as determining the probability of the unambiguous identity of the amino acid. For example, in some embodiments, an amino acid is identified by determining the probability (e.g., 0% to 100%) that the amino acid is a particular type, or by determining the probability of each of a plurality of particular types. Thus, in some embodiments, the terms "amino acid sequence," "polypeptide sequence," and "protein sequence" as used herein can refer to the polypeptide or protein substance itself, and are not limited to the specific sequence information that biochemically characterizes a particular polypeptide or protein (e.g., a series of letters representing the order of amino acids from one end to the other).

[0244] In some embodiments, sequencing the polypeptide molecule comprises identifying at least two (e.g., at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, or more) amino acids in the polypeptide molecule. In some embodiments, the at least two amino acids are consecutive amino acids. In some embodiments, the at least two amino acids are non-consecutive amino acids.

[0245] In some embodiments, the sequencing of the polypeptide molecule includes identification of less than 100% of all amino acids in the polypeptide molecule (e.g., less than 99%, less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 1%, or less). For example, in some embodiments, the sequencing of the polypeptide molecule includes identification of less than 100% of one type of amino acid in the polypeptide molecule (e.g., identification of a portion of all amino acids of one type in the polypeptide molecule). In some embodiments, the sequencing of the polypeptide molecule includes identification of less than 100% of each type of amino acid in the polypeptide molecule.

[0246] In some embodiments, sequencing of a polypeptide molecule involves identification of at least 1, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, or more types of amino acids in the polypeptide.

[0247] In some embodiments, the present application provides compositions and methods for sequencing a polypeptide by identifying a series of amino acids present at the terminus of the polypeptide over time (e.g., by repeated detection and cleavage of terminal amino acids). In yet other embodiments, the present application provides compositions and methods for sequencing a polypeptide by identifying the labeled amino content of the polypeptide and comparing it to a reference sequence database.

[0248] In some embodiments, the present application provides compositions and methods for sequencing a polypeptide by sequencing multiple fragments of the polypeptide. In some embodiments, sequencing the polypeptide comprises combining sequence information of multiple polypeptide fragments to identify and / or determine the sequence of the polypeptide. In some embodiments, combining sequence information may be performed by computer hardware and software. The methods described herein may allow for sequencing a set of related polypeptides, for example, the entire proteome of an organism. In some embodiments, multiple single molecule sequencing reactions are performed in parallel (e.g., on a single chip) according to aspects of the present application. For example, in some embodiments, multiple single molecule sequencing reactions are each performed in separate sample wells on a single chip.

[0249] In some embodiments, the methods provided herein may be used to sequence and identify individual proteins in a sample containing a complex mixture of proteins. In some embodiments, the present application provides a method for uniquely identifying individual proteins in a complex mixture of proteins. In some embodiments, individual proteins are detected in a mixed sample by determining a partial amino acid sequence of the protein. In some embodiments, the partial amino acid sequence of the protein is within a contiguous stretch of approximately 5-50 amino acids.

[0250] Without wishing to be bound by any particular theory, it is believed that most human proteins can be identified using incomplete sequence information by reference to proteomic databases. For example, simple modeling of the human proteome has shown that approximately 98% of proteins can be uniquely identified by detecting just four types of amino acids within a stretch of 6-40 amino acids (see, for example, Swaminathan et al., PLoS Comput Biol. 2015, vol. 11, no. 2, p. e1004080; and Yao et al., Phys. Biol. 2015, vol. 12, no. 5, p. 055003). Thus, a complex mixture of proteins can be degraded (e.g., chemically, enzymatically) into short polypeptide fragments of approximately 6-40 amino acids, and sequencing of this polypeptide library will reveal the identity and abundance of each of the proteins present in the original complex mixture. Compositions and methods for selective amino acid labeling and identification of polypeptides by determining partial sequence information are described in detail in U.S. Patent Application Serial No. 15 / 510,962, filed September 15, 2015, entitled "SINGLE MOLECULE PEPTIDE SEQUENCING," which is incorporated by reference in its entirety.

[0251] Embodiments can sequence single polypeptide molecules with high accuracy, such as at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99%, 99.999%, or 99.9999% accuracy. In some embodiments, the target molecule used for single molecule sequencing is a polypeptide immobilized on a surface of a solid support, such as a bottom surface or a sidewall surface of a sample well. The sample well can also contain any other reagents required for the sequencing reaction of the present application, such as one or more suitable buffers, cofactors, labeled affinity reagents, and enzymes (e.g., catalytically active or inactive exopeptidase enzymes, which can be luminescently labeled or unlabeled).

[0252] As described above, in some embodiments, the present sequencing involves identifying amino acids by determining the probability that the amino acid is of a particular type. Conventional protein identification systems require the identification of each amino acid in a polypeptide in order to identify the polypeptide. However, it is difficult to accurately identify each amino acid in a polypeptide. For example, data collected from an interaction in which a first recognition molecule associates with a first amino acid may not differ enough from data collected from an interaction in which a second recognition molecule associates with a second amino acid to distinguish between the two amino acids. In some embodiments, the present sequencing circumvents this problem by using a protein identification system that does not require (but does not preclude) the identification of each amino acid in a protein, unlike conventional protein identification systems.

[0253] Thus, in some embodiments, sequencing according to the present application may be performed using a protein identification system that uses machine learning techniques to identify proteins. In some embodiments, the system operates by (1) collecting data about a protein polypeptide using a real-time protein sequencing device, (2) identifying the probability that a particular amino acid is part of the polypeptide at each position using a machine learning model and the collected data, and (3) identifying the protein using the identification probability as a "probabilistic fingerprint". In some embodiments, data about a protein polypeptide may be obtained using a reagent that selectively binds to an amino acid. By way of example, the reagent and / or the amino acid may be labeled with a luminescent label that emits light in response to the application of excitation energy. In this example, the protein sequencing device may apply excitation energy to a sample of a protein (e.g., a polypeptide) upon binding interaction between the reagent and an amino acid in the sample. In some embodiments, one or more sensors (e.g., a photodetector, an electrical sensor, and / or any other suitable type of sensor) of the sequencing device may detect upon binding interaction. Meanwhile, data collected and / or obtained from the detected light emission may be provided to a machine learning model. The machine learning models and related systems and methods are described in detail in U.S. Provisional Patent Application No. 62 / 860,750, filed June 12, 2019, entitled "MACHINE LEARNING ENABLED PROTEIN IDENTIFICATION," which is incorporated by reference in its entirety.

[0254] Sequencing according to the present application may, in some aspects, include immobilizing a polypeptide on a surface of a substrate (e.g., a solid support, e.g., a chip, e.g., an integrated device as described herein). In some embodiments, the polypeptide may be immobilized on a surface of a sample well on the substrate (e.g., on the bottom surface of the sample well). In some embodiments, the N-terminal amino acid of the polypeptide is immobilized (e.g., attached to the surface). In some embodiments, the C-terminal amino acid of the polypeptide is immobilized (e.g., attached to the surface). In some embodiments, one or more non-terminal amino acids are immobilized (e.g., attached to the surface). The immobilized amino acids can be attached using any suitable covalent or non-covalent linkage, e.g., as described herein. In some embodiments, multiple polypeptides are attached to multiple sample wells, e.g., in an array of sample wells on the substrate (e.g., one polypeptide is attached to the surface, e.g., the bottom surface, of each sample well).

[0255] The sequencing according to the present application may, in some aspects, be performed using a system that allows single molecule analysis. The system may include an integrating device and an instrument configured to interface with the integrating device. The integrating device may include an array of pixels, and each pixel may include a sample well and at least one photodetector. The sample wells of the integrating device may be formed on or across the surface of the integrating device and may be configured to receive a sample disposed on the surface of the integrating device. Collectively, the sample wells may be considered an array of sample wells. The multiple sample wells may have a suitable size and shape such that at least a portion of the sample wells receive a single sample (e.g., a single molecule such as a polypeptide). In some embodiments, the number of samples in the sample wells may be distributed among the sample wells of the integrating device such that some sample wells contain one sample, while others contain zero, two, or more samples.

[0256] Excitation light is provided to the integrating device from one or more light sources external to the integrating device. An optical component of the integrating device may receive the excitation light from the light source, direct the light to the array of sample wells of the integrating device, and illuminate an illumination area within the sample well. In some embodiments, the sample wells may have a configuration that allows a sample to be held in close proximity to a surface of the sample well and facilitates delivery of excitation light to the sample and detection of emission light from the sample. A sample positioned within the illumination area may emit emission light in response to being illuminated by the excitation light. For example, the sample may be labeled with a fluorescent marker that emits light in response to achieving an excited state via illumination with the excitation light. The emission light emitted by the sample may then be detected by one or more photodetectors in pixels that correspond to the sample wells having the sample to be analyzed. Multiple samples can be analyzed in parallel when performed across an array of sample wells, which according to some embodiments may be in the range of approximately 10,000 pixels to 1,000,000 pixels in number.

[0257] The integrating device may include an optical system for receiving excitation light and directing the excitation light between the sample well arrays. The optical system may include one or more grating couplers configured to couple the excitation light to the integrating device and direct the excitation light to other optical components. The optical system may include optical components that direct the excitation light from the grating couplers toward the sample well arrays. Such optical components may include an optical splitter, an optical combiner, and a waveguide. In some embodiments, the one or more optical splitters may combine the excitation light from the grating couplers and deliver the excitation light to at least one of the waveguides. According to some embodiments, the optical splitter may have a configuration that allows for a substantially uniform delivery of excitation light across all of the waveguides such that each of the waveguides receives a substantially similar amount of excitation light. Such embodiments may improve the performance of the integrating device by improving the uniformity of the excitation light received by the sample wells of the integrating device. For example, examples of components suitable for inclusion in an integrated device for coupling excitation light to a sample well and / or directing emission light to a photodetector are described in U.S. patent application Ser. No. 14 / 821,688, filed Aug. 7, 2015, entitled "INTEGRATED DEVICE FOR PROBING, DETECTING AND ANALYZING MOLECULES," and U.S. patent application Ser. No. 14 / 543,865, filed Nov. 17, 2014, entitled "INTEGRATED DEVICE WITH EXTERNAL LIGHT SOURCE FOR PROBING, DETECTING, AND ANALYZING MOLECULES," both of which are incorporated by reference in their entireties.Examples of suitable grating couplers and waveguides that may be implemented in the integrated device are described in U.S. patent application Ser. No. 15 / 844,403, filed Dec. 15, 2017, entitled “OPTICAL COUPLER AND WAVEGUIDE SYSTEM,” which is incorporated by reference in its entirety.

[0258] Additional photonic structures may be positioned between the sample well and the photodetector and may be configured to reduce or prevent excitation light from reaching the photodetector that may contribute to signal noise when detecting emission light. In some embodiments, the metal layer that may act as the circuitry of the integrated device may also act as a spatial filter. Examples of suitable photonic structures may include spectral filters, polarizing filters, and spatial filters, and are described in U.S. Patent Application No. 16 / 042,968, filed July 23, 2018, entitled "OPTICAL REJECTION PHOTONIC STRUCTURES," which is incorporated by reference in its entirety.

[0259] Components positioned remotely from the integrating device may be used to position and align the excitation source to the integrating device. Such components may include optical components, including lenses, mirrors, prisms, windows, apertures, attenuators, and / or optical fibers. Additional mechanical components may be included in the instrument to allow control of one or more alignment components. Such mechanical components may include actuators, stepper motors, and / or knobs. Examples of suitable excitation sources and alignment mechanisms are described in U.S. Patent Application Serial No. 15 / 161,088, filed May 20, 2016, entitled "PULSED LASER AND SYSTEM," which is incorporated by reference in its entirety. Another example of a beam steering module is described in U.S. Patent Application No. 15 / 842,720, filed December 14, 2017, entitled "COMPACT BEAM SHAPING AND STEERING ASSEMBLY," which is incorporated herein by reference. Additional examples of suitable excitation sources are described in U.S. Patent Application No. 14 / 821,688, filed August 7, 2015, entitled "INTEGRATED DEVICE FOR PROBING, DETECTING AND ANALYZING MOLECULES," which is incorporated herein by reference in its entirety.

[0260] Photodetectors positioned with individual pixels of the integrating device may be configured and positioned to detect emitted light from the pixel's corresponding sample well. Examples of suitable photodetectors are described in U.S. Patent Application No. 14 / 821,656, filed August 7, 2015, entitled "INTEGRATED DEVICE FOR TEMPORAL BINNING OF RECEIVED PHOTONS," which is incorporated by reference in its entirety. In some embodiments, the sample wells and their respective photodetectors may be aligned along a common axis. In this way, the photodetectors may overlap the sample wells within a pixel.

[0261] A characteristic of the detected emission light may provide an indication for identifying a marker associated with the emission light. Such characteristics may include any suitable type of characteristic, including the arrival time of a photon detected by a photodetector, the amount of photons accumulated over time by a photodetector, and / or the distribution of photons across two or more photodetectors. In some embodiments, the photodetector may have a configuration that allows detection of one or more timing characteristics (e.g., luminescence lifetime) associated with the emission light of the sample. The photodetector may detect a distribution of photon arrival times after a pulse of excitation light propagates through the integrating device, and the distribution of arrival times may provide an indication of the timing characteristics of the emission light of the sample (e.g., a proxy for the luminescence lifetime). In some embodiments, the one or more photodetectors provide an indication of the probability of the emission light being emitted by the marker (e.g., luminescence intensity). In some embodiments, the multiple photodetectors may be sized and positioned to capture the spatial distribution of the emission light. The output signal from the one or more photodetectors may then be used to distinguish the marker among the multiple markers, and the multiple markers may be used to identify the sample in the sample. In some embodiments, a sample may be excited with multiple excitation energies, and emission light and / or timing characteristics of the emission light emitted by the sample in response to the multiple excitation energies may distinguish a marker from multiple markers.

[0262] In operation, parallel analysis of samples in the sample wells is performed by exciting some or all of the samples in the wells with excitation light and detecting signals from the sample emissions with a photodetector. Emission light from the samples can be detected by corresponding photodetectors and converted into at least one electrical signal. The electrical signal can be transmitted along conductive lines in the circuitry of the integrated device and can be connected to an instrument interfaced to the integrated device. The electrical signal can be further processed and / or analyzed. Processing or analysis of the electrical signal can be performed on a suitable computing device located either on the instrument or off the instrument.

[0263] The appliance may include a user interface for controlling the operation of the appliance and / or an integrated device. The user interface may be configured to allow a user to input information into the appliance, such as commands and / or settings used to control the functionality of the appliance. In some embodiments, the user interface may include buttons, switches, dials, and a microphone for voice commands. The user interface may allow a user to receive feedback regarding the performance of the appliance and / or an integrated device, such as information obtained by proper alignment and / or a readout signal from a light detector on the integrated device. In some embodiments, the user interface may provide feedback using a speaker to provide audible feedback. In some embodiments, the user interface may include indicator lights and / or a display screen to provide visual feedback to the user.

[0264] In some embodiments, the instrument may include a computer interface configured to connect to a computing device. The computer interface may be a USB interface, a FireWire™ interface, or any other suitable computer interface. The computing device may be any general-purpose computer, such as a laptop computer or a desktop computer. In some embodiments, the computing device may be a server (e.g., a cloud-based server) accessible over a wireless network via a suitable computer interface. The computer interface may facilitate communication of information between the instrument and the computing device. Input information for controlling and / or configuring the instrument may be provided to the computing device and communicated to the instrument via the computer interface. Output information generated by the instrument may be received by the computing device via the computer interface. The output information may include feedback regarding the performance of the instrument, performance of the integrated device, and / or data generated from a readout signal of a photodetector.

[0265] In some embodiments, the instrument may include a processing device configured to analyze data received from one or more photodetectors of the integrated device and / or to transmit control signals to the excitation source. In some embodiments, the processing device may include a general-purpose processor, a specially-adapted processor (e.g., a central processing unit (CPU), e.g., one or more microprocessor or microcontroller cores, a field programmable gate array (FPGA), an application specific integrated circuit (ASIC), a custom integrated circuit, a digital signal processor (DSP), or a combination thereof). In some embodiments, processing of data from one or more photodetectors may be performed by both the instrument's processing device and an external computing device. In other embodiments, the external computing device may be omitted and processing of data from one or more photodetectors may be performed only by the processing device of the integrated device.

[0266] According to some embodiments, an instrument configured to analyze a sample based on luminescence emission characteristics may detect differences in luminescence lifetimes and / or intensities between different luminescent molecules and / or the lifetimes and / or intensities of the same luminescent molecule in different environments. The inventors have recognized and appreciated that differences in luminescence emission lifetimes can be used to distinguish the presence or absence of different luminescent molecules and / or to distinguish different environments or conditions to which the luminescent molecules are exposed. In some cases, distinguishing luminescent molecules based on lifetimes (e.g., rather than emission wavelengths) may simplify aspects of the system. By way of example, wavelength-distinguishing optical elements (e.g., wavelength filters, dedicated detectors for each wavelength, dedicated pulsed light sources for different wavelengths, and / or diffractive optical elements) may be reduced in number or eliminated when distinguishing luminescent molecules based on lifetimes. In some cases, a single pulsed light source operating at a single characteristic wavelength may be used to excite different luminescent molecules that emit within the same wavelength region of the optical spectrum but have measurably different lifetimes. Analysis systems that use a single pulsed light source, rather than multiple light sources operating at different wavelengths, to excite and distinguish different luminescent molecules that emit in the same wavelength region can be less complicated to operate and maintain, more compact, and less costly to manufacture.

[0267] Although an analysis system based on luminescence lifetime analysis may have certain advantages, the amount of information and / or detection accuracy obtained by the analysis system may be increased by allowing additional detection techniques. For example, some embodiments of the system may be additionally configured to distinguish one or more properties of the sample based on the emission wavelength and / or the emission intensity. In some implementations, the emission intensity may additionally or alternatively be used to distinguish different luminescent labels. For example, some luminescent labels may emit at significantly different intensities or have significant differences in their excitation probabilities (e.g., at least about 35% difference) even though their decay rates may be similar. By referencing the bin signal to the measured excitation light, it may be possible to distinguish different luminescent labels based on intensity levels.

[0268] According to some embodiments, different luminescence lifetimes can be distinguished using a photodetector configured to time-bin luminescence emission events following excitation of the luminescent label. Time binning can occur during a single charge accumulation cycle for the photodetector. A charge accumulation cycle is an interval between readout events during which photogenerated carriers are accumulated in bins of the time binning photodetector. An example of a time binning photodetector is described in U.S. Patent Application No. 14 / 821,656, filed August 7, 2015, entitled "INTEGRATED DEVICE FOR TEMPORAL BINNING OF RECEIVED PHOTONS," which is incorporated herein by reference. In some embodiments, the time binning photodetector can generate charge carriers in a photon absorption / carrier generation region and directly transfer the charge carriers to charge carrier storage bins in a charge carrier storage region. In such embodiments, the time binning photodetector may not include a carrier transfer / capture region. Such time binning photodetectors may be referred to as “direct binning pixels.” Examples of time binning photodetectors that include direct binning pixels are described in U.S. Patent Application No. 15 / 852,571, filed December 22, 2017, entitled “INTEGRATED PHOTODETECTOR WITH DIRECT BINNING PIXEL,” which is incorporated herein by reference.

[0269] In some embodiments, different numbers of fluorophores of the same type can be linked to different reagents in a sample, so that each reagent can be identified based on emission intensity. For example, two fluorophores can be linked to a first labeled affinity reagent, and four or more fluorophores can be linked to a second labeled affinity reagent. Because of the different numbers of fluorophores, there can be different excitation probabilities and fluorophore emission probabilities associated with the different affinity reagents. For example, there can be more emission events during a signal accumulation interval in the second labeled affinity reagent, so that the apparent intensity of the bin is significantly higher than that of the first labeled affinity reagent.

[0270] The inventors have recognized and appreciated that distinguishing nucleotides or any other biological or chemical samples based on fluorophore decay rates and / or fluorophore intensity may allow for simplification of optical excitation and detection systems. For example, optical excitation may be performed with a single wavelength light source (e.g., a light source that produces one characteristic wavelength rather than multiple light sources or a light source that operates at multiple different characteristic wavelengths). Alternatively, wavelength-discriminating optics and filters may not be required for the detection system. Also, a single photodetector may be used for each sample well to detect emissions from different fluorophores. The phrase "characteristic wavelength" or "wavelength" is used to mean the central or dominant wavelength within a limited bandwidth of radiation (e.g., the central or peak wavelength within a 20 nm bandwidth output by a pulsed light source). In some cases, "characteristic wavelength" or "wavelength" may be used to mean the peak wavelength within the total bandwidth of radiation output by the light source.

[0271] calculation technology Aspects of the present application relate to computational techniques for analyzing data generated by the polypeptide sequencing techniques described herein. As discussed above, for example, in relation to Figures 1A and 1B, data generated using these sequencing techniques may include a series of signal pulses indicative of instances in which an amino acid recognition molecule associates with an amino acid exposed at the end of the polypeptide being sequenced. The series of signal pulses may have one or more different features (e.g., pulse duration, inter-pulse duration, change in magnitude) depending on the type of amino acid at the current end over time as the degradation process proceeds by removing successive amino acids. The resulting signal trace may include characteristic patterns resulting from one or more different features associated with each amino acid. The computational techniques described herein may be implemented as part of analyzing such data obtained using these sequencing techniques to identify amino acid sequences.

[0272] Some embodiments may include obtaining data during a degradation process of a polypeptide, analyzing the data to determine portions of the data that correspond to amino acids that are sequentially exposed at the ends of the polypeptide during the degradation process, and outputting an amino acid sequence corresponding to the polypeptide. Figure 11 is a diagram of an exemplary processing pipeline 1100 for identifying an amino acid sequence by analyzing data obtained using the polypeptide sequencing techniques described herein. As shown in Figure 11, analyzing the sequencing data 1102 may include using an association event identification technique 1104 and an amino acid identification technique 1106 to output an amino acid sequence 1108.

[0273] As discussed herein, sequencing data 1102 can be obtained during a degradation process of a polypeptide. In some embodiments, the sequencing data 1102 is indicative of the amino acid identity of the termini of the polypeptide during the degradation process. In some embodiments, the sequencing data 1102 is indicative of signals generated by one or more amino acid recognition molecules that bind to different types of terminal amino acids at the termini during the degradation process. Exemplary sequencing data is shown in Figures 1A and 1B, discussed above.

[0274] Depending on how the signal is generated during the degradation process, the sequencing data 1102 can be indicative of one or more different types of signals. In some embodiments, the sequencing data 1102 is indicative of a luminescent signal generated during the degradation process. For example, a luminescent label can be used to label an amino acid recognition molecule, and luminescence emitted by the luminescent label can be detected when the amino acid recognition molecule associates with a particular amino acid resulting in a luminescent signal. In some embodiments, the sequencing data 1102 is indicative of an electrical signal generated during the degradation process. For example, a polypeptide molecule to be sequenced can be immobilized in a nanopore, and an electrical signal (e.g., a change in conductance) can be detected when the amino acid recognition molecule associates with a particular amino acid.

[0275] Some embodiments include analyzing the sequencing data 1102 to determine portions of the sequencing data 1102 that correspond to amino acids that are sequentially exposed at the ends of the polypeptide during the degradation process. As shown in FIG. 11, an association event identification technique 1104 may access the sequencing data 1102 and analyze the sequencing data to identify portions of the sequencing data 1102 that correspond to association events. Association events are identified in the data as CPs shown in FIG. 1B. 1 Or CP 2In some embodiments, the association event identification technique 1104 may include detecting a series of cleavage events and determining the portion of the sequencing data 1102 between the successive cleavage events. As an example, the CP 1 and CP 2 The disconnection event between CP 1 A first portion of the data corresponding to the first meeting event may be identified as a first meeting event, and CP 2 can be detected such that a second portion of the data corresponding to can be identified as a second association event.

[0276] Some embodiments include identifying a type of amino acid for one or more of the determined portions of the sequencing data 1102. As shown in FIG. 11, an amino acid identification technique 1106 may be used to determine a type of amino acid for one or more of the association events identified by the association event identification technique 1104. In some embodiments, the individual portions of data identified by the association event identification technique 1104 may include pulse patterns, and the amino acid identification technique 1106 may determine a type of amino acid for the one or more portions based on their respective pulse patterns. With reference to FIG. 1B, the amino acid identification technique 1106 may determine a type of amino acid for one or more of the determined portions of the sequencing data 1102. 1 1st type amino acid and CP 2 In some embodiments, determining the type of amino acid may include identifying a quantity of time within a portion of the data, such as a portion identified using association event identification technique 1104 when the data is above a threshold, and comparing the quantity of time and duration for the portion of the data. For example, CP 1 Identifying the type of amino acid for CP when the signal is above threshold 1 The amount of time within, say, the signal is L Determine the time period pd when CP 1and comparing it to the total duration. In some embodiments, determining the type of amino acid may include identifying one or more pulse durations for one or more portions of the data identified by the association event identification technique 1102. For example, CP 1 Identifying the type of amino acid for CP 1 In some embodiments, determining the type of amino acid may include identifying one or more inter-pulse durations for one or more portions of the data identified using the association event identification technique 1104. For example, CP 1 Identifying the type of amino acid for may include identifying an inter-pulse duration, such as ipd.

[0277] By identifying types of amino acids for sequential portions of the sequencing data 1102, the amino acid identification technique 1106 may output an amino acid sequence 1108 that corresponds to the polypeptide. In some embodiments, the amino acid sequence comprises a series of amino acids that correspond to portions of the data identified using the association event identification technique 1104.

[0278] 12 is a flow chart of an exemplary process 1200 for determining an amino acid sequence of a polypeptide molecule according to some embodiments of the technology described herein. Process 1200 may be implemented on any suitable computing device (e.g., a single computing device, multiple computing devices co-located at a single physical location or located at multiple physical locations remote from each other, one or more computing devices part of a cloud computing system, etc.), as aspects of the technology described herein are not limited in this respect. In some embodiments, association event identification technology 1104 and amino acid identification technology 1106 may implement some or all of process 1200 to determine the amino acid sequence.

[0279] Process 1200 begins with operation 1202, which involves contacting a single polypeptide molecule with one or more terminal amino acid recognition molecules. Process 1200 then proceeds to operation 1104, which involves detecting a series of signal pulses indicative of association of one or more terminal amino acid recognition molecules with sequential amino acids exposed at the termini of the single polypeptide as the single polypeptide is degraded. The series of pulses may enable sequencing of the single polypeptide molecule, for example, by using association event identification techniques 1104 and amino acid identification techniques 1106.

[0280] In some embodiments, process 1200 may include operation 1206 including identifying a first type of amino acid of the single polypeptide molecule based on a first characteristic pattern of the series of signal pulses, for example, by using amino acid identification technique 1106.

[0281] 13 is a flow chart of an exemplary process 1300 for determining an amino acid sequence corresponding to a polypeptide according to some embodiments of the technology described herein. Process 1300 may be implemented on any suitable computing device (e.g., a single computing device, multiple computing devices co-located at a single physical location or located at multiple physical locations remote from each other, one or more computing devices part of a cloud computing system, etc.), as aspects of the technology described herein are not limited in this respect. In some embodiments, association event identification technology 1104 and amino acid identification technology 1106 may implement some or all of process 1300 to determine the amino acid sequence.

[0282] Process 1300 begins at operation 1302 where data is obtained during a degradation process of a polypeptide. In some embodiments, the data is indicative of amino acid identity at the termini of the polypeptide during the degradation process. In some embodiments, the data is indicative of signals generated by one or more amino acid recognition molecules that bind to different types of terminal amino acids at the termini during the degradation process. In some embodiments, the data is indicative of luminescent signals generated during the degradation process. In some embodiments, the data is indicative of electrical signals generated during the degradation process.

[0283] Process 1300 then proceeds to operation 1304 where the data is analyzed to determine portions of the data that correspond to amino acids that are sequentially exposed at the termini of the polypeptide during the degradation process, for example, by using association event identification techniques 1104 and amino acid identification techniques 1106. In some embodiments, analyzing the data further includes detecting a series of cleavage events and determining portions of the data between the sequential cleavage events, for example, by using association event identification techniques 1104.

[0284] In some embodiments, analyzing the data further includes determining a type of amino acid for each of the discrete portions, for example, by using amino acid identification techniques 1106. In some embodiments, each of the discrete portions includes a pulse pattern, and analyzing the data further includes determining a type of amino acid for one or more portions based on its respective pulse pattern. In some embodiments, determining the type of amino acid further includes identifying an amount of time within the portion when the data is above a threshold, and comparing the amount of time and duration for the portion. In some embodiments, determining the type of amino acid further includes identifying at least one pulse duration for each of the one or more portions. In some embodiments, determining the type of amino acid further includes identifying at least one inter-pulse duration for each of the one or more portions.

[0285] Process 1300 then proceeds to operation 1306 where an amino acid sequence corresponding to the polypeptide is output, for example via a user interface. In some embodiments, the amino acid sequence includes a series of amino acids corresponding to the moiety.

[0286] An exemplary implementation of a computer system 1400 that may be used in conjunction with any of the embodiments of the technology described herein is illustrated in FIG. 14. The computer system 1400 includes one or more processors 1410 and one or more articles of manufacture that include a non-transitory computer-readable storage medium (e.g., memory 1420 and one or more non-volatile storage media 1430). The processor 1410 may control data writing and reading to the memory 1420 and the non-volatile storage device 1430 in any suitable manner, although aspects of the technology described herein are not limited in this respect. To perform any of the functionality described herein, the processor 1410 may execute one or more processor-executable instructions stored in one or more non-transitory computer-readable storage media (e.g., memory 1420), which may function as a non-transitory computer-readable storage medium that stores processor-executable instructions executed by the processor 1410.

[0287] Computing device 1400 may also include a network input / output (I / O) interface 1440 that may mediate communications between the computing device and other computing devices (e.g., over a network), as well as one or more user I / O interfaces 1450 that may mediate the computing device providing output to and receiving input from a user. User I / O interfaces may include devices such as a keyboard, a mouse, a microphone, a display device (e.g., a monitor or touch screen), speakers, a camera, and / or various other types of I / O devices.

[0288] The above described embodiments can be implemented in any of many ways. For example, the embodiments can be implemented using hardware, software, or a combination thereof. When implemented in software, the software code can be executed on any suitable processor (e.g., a microprocessor) or group of processors, whether provided on a single computing device or distributed across multiple computing devices. It should be appreciated that any component or group of components implementing the above described functions can be generally considered to be one or more controllers that control the functions discussed above. The one or more controllers can be implemented in many ways, for example, using dedicated hardware or general purpose hardware (e.g., one or more processors) programmed with microcode or software to implement the functions described above.

[0289] In this regard, it should be appreciated that an implementation of the embodiments described herein includes at least one computer-readable storage medium (e.g., RAM, ROM, EEPROM, flash memory, or other memory technology, CD-ROM, digital versatile disk (DVD), or other optical disk storage, magnetic cassette, magnetic tape, magnetic disk storage, or other magnetic storage device, or other tangible non-transitory computer-readable storage medium) coded with a computer program (i.e., a plurality of executable instructions) that, when executed on one or more processors, performs the functions discussed above of one or more embodiments. The computer-readable medium may be transportable such that the stored program may be loaded into any computing device to implement aspects of the technology discussed herein. Additionally, it should be appreciated that references to a computer program that, when executed, performs any of the functions discussed above are not limited to application programs running on a host computer. Rather, the terms computer program and software are used herein in a generic sense to refer to any type of computer code (e.g., application software, firmware, microcode, or any other form of computer instructions) available to program one or more processors to implement aspects of the technology discussed herein. EXAMPLES

[0290] Example 1. Edman Degradation by Chemical Cleavage

[0291] [ka]

[0292] Surface-mounted oligopeptides of about 3 to about 30 amino acids (n=3-30) are provided, where R 1 ~R 3can be any of the 20 common amino acids or endogenously modified amino acids (e.g., modified by post-translational modification). In step 1 of the isothiocyanate N-terminal reaction, an isothiocyanate X-NCS is added to a vessel containing a surface-mounted oligopeptide, where X is phenyl (Ph), 4-NO 2 Ph, 4-SO 3 The thiourea cleavage reaction is carried out under the following conditions: aqueous buffer pH 4-10, MeOH or EtOH or IPA alcoholic co-solvent, trialkylamine in organic solvent (DCM, THF, MeCN, DMF, etc.), 20°C-50°C to give the X-NCS derivatized N-terminal amino acid. In step 2 of the thiourea cleavage reaction, an acid or base is added to the vessel containing the X-NCS derivatized N-terminal amino acid, where the acid is acetic acid, formic acid, trichloroacetic acid, trifluoroacetic acid, phosphoric acid, or hydrochloric acid, either neat or in aqueous solution, or the base is trialkylamine or buffered trialkylamine (e.g., EtOH, EtOH, IPA, etc.). 3 NH + AcO - Step 2 is carried out under the following conditions: neat acid or any ratio of aqueous / organic co-solvents at 20° C. to 50° C. to give the n-1 oligopeptide and a thiohydantoin by-product.

[0293] Example 2. Solubilizing linkers for peptide surface immobilization In an attempt to improve the solubility of oligopeptides in aqueous buffers, it was determined that conjugating peptide fragments to oligonucleotide linkers could improve aqueous solubility while also providing a functional moiety for surface immobilization of the peptide at the single molecule level. A variety of peptide-linker conjugates were synthesized, and example structures are depicted in Figure 15A for peptide-DNA and peptide-PEG conjugates. Linker conjugation was observed to greatly enhance the solubility of the peptide in aqueous solutions for each of the various peptide-linker conjugates evaluated.

[0294] The peptide-linker conjugates were evaluated for amino acid cleavage at the peptide N-terminus by N-terminal aminopeptidase (Table 6 below).

[0295] [Table 6]

[0296] The peptide-linker conjugates shown in Table 6 were incubated with either proline iminopeptidase ("PIP") or rat aminopeptidase N ("rat APN"), and peptide cleavage was monitored by LCMS. An example of LCMS demonstrating the cleavage of entry 5 of Table 6 is shown in Figure 15B. All other cleavage reactions were measured similarly. As shown in Table 6, the positively charged peptide-DNA conjugates ("oligo" and "oligo-PEG" linkers) were not cleaved by the tested aminopeptidases, while all other conjugate classes (negatively charged, aromatic, hydrophobic) with DNA oligonucleotide linkers were cleaved. Comparison showed that the positively charged peptide-PEG conjugates were cleaved by at least one of the aminopeptidases.

[0297] It has been shown that peptides of different compositions can be immobilized on individual sample well surfaces for single molecule analysis using labeled peptide-linker conjugates. In these experiments, the DNA linker was labeled with a dye (e.g., as depicted in FIG. 15A for peptide-DNA conjugates), and the loading of different peptide-DNA conjugates into individual sample wells was measured by dye fluorescence. An example loading experiment is shown in FIG. 15C. By measuring the fluorescence emission of labeled peptide-DNA conjugates (50 pM), it was determined that at least 18% of the sample wells on the chip were loaded with a surface-immobilized conjugate in a single occupancy per sample well. These experiments demonstrated that peptide-linker conjugates exhibit enhanced aqueous solubility compared to unconjugated peptide counterparts, that the conjugated linkers do not prevent terminal amino acid cleavage of peptides by different aminopeptidases, and that peptide-linker conjugates of different compositions can be immobilized on the chip surface at the single molecule level.

[0298] Example 3. Exopeptidase cleavage of a polypeptide substrate The cleavage ability of various aminopeptidases was tested. The conditions and results of a set of cleavage assay experiments, including peptide substrate concentration, enzyme concentration, buffer conditions, temperature, and incubation time, are shown in Table 7. High performance liquid chromatography (HPLC) was used to assay cleavage of the peptide substrates by the indicated enzymes. The "HPLC Assay Conversion" values ​​in Table 7 represent the percent of the peptide substrate that was converted to cleavage products. To determine the "HPLC Assay Conversion" values, two solutions containing the same starting concentration of peptide were prepared. One solution was subjected to enzymatic digestion, while the other solution did not contain any enzyme but was diluted with an equivalent amount of buffer used to store the enzymes. The reaction was quenched at the indicated times. The amount of reactant converted to product was determined by dividing the HPLC peak area of ​​the starting material remaining after enzymatic digestion by the peak area of ​​a control solution of undigested peptide and then multiplying this ratio by 100. In Table 7, "NH2" represents an amine group, "yPIP" stands for Y. pestis proline iminopeptidase, "NPEPPS" stands for puromycin-sensitive aminopeptidase, "VPr" stands for Vibrio proteolyticus aminopeptidase, and "EDAPN" stands for L. pneumophila M1 aminopeptidase.

[0299] [Table 7-1]

[0300] [Table 7-2]

[0301] [Table 7-3]

[0302] [Table 7-4]

[0303]

Table 7-5

[0304]

Table 7-6

[0305]

Table 7-7

[0306]

Table 7-8

[0307]

Table 7-9

[0308]

Table 7-10

[0309]

Table 7-11

[0310]

Table 7-12

[0311]

Table 7-13

[0312]

Table 7-14

[0313] A summary of the amino acid cleavage activity of selected exopeptidases in Table 7 is shown in Figure 16. Specific cleavage activities are shown for the following enzymes: "cVPr" (V. proteolyticus aminopeptidase), "yPIP" (Y. pestis proline iminopeptidase), "D / E APN" (L. pneumophila M1 aminopeptidase), hTET (Pyrococcus horikoshii TET aminopeptidase), and Pfu API ("PfuTET" in Table 7). Specific activity against terminal amino acids is categorized as indicated, using the single letter abbreviation for the amino acid ("XP-" represents any terminal amino acid (X) with an adjacent or penultimate proline (P) residue).

[0314] Example 4. Terminal amino acid truncation of immobilized peptides at the single molecule level An assay for on-chip amino acid cleavage of immobilized peptides was developed using labeled peptide conjugates. The assay was designed to provide a method to determine the enzymatic recognition and cleavage activity of exopeptidases towards immobilized peptides. This may allow the measurement of kinetic binding parameters and general binding affinities.

[0315] To assess N-terminal amino acid cleavage of peptides, we designed and synthesized dye-labeled peptides containing an N-terminal aspartic acid attached to the dye via a PEG spacer. The peptides also contained a proline residue adjacent to a modified aspartic acid that is specifically recognized by the enzyme proline iminopeptidase (derived from Yersinia pestis and known elsewhere, referred to herein as "yPIP"). The enzyme yPIP should cleave only the N-terminal amino acid upstream of the proline residue.

[0316] After showing that this and other labeled peptides are efficiently cleaved by yPIP in bulk (e.g., as described in Example 2), we developed an on-chip dye / peptide conjugate assay to monitor N-terminal amino acid cleavage at the single molecule level. Figure 17A shows a general scheme of the dye / peptide conjugate assay (insert panel). As shown, a peptide with a label attached to its N-terminal amino acid via a spacer is immobilized to the surface via a linker. After exposure to a peptidase, N-terminal amino acid cleavage results in the removal of the labeled residue from the detectable observation volume and is measured by the concomitant loss of signal from the label. The enzyme-peptide complex on the right of the inset panel generally depicts the N-terminal cleavage site.

[0317] Figure 17A shows labeled peptide constructs designed and synthesized for use in dye / peptide conjugate assays (bottom). In these experiments, a rhodamine dye (ATTO Rho6G) was attached to the N-terminal aspartic acid residue of a peptide bearing a penultimate proline residue at the N-terminus. As indicated, the peptide was further conjugated to a solubilizing DNA linker bearing a biotin moiety for surface immobilization.

[0318] The labeled peptide conjugates were loaded onto a glass chip with an array of sample wells. Images of the chip were acquired before and after loading to determine the percent loading of single-occupied sample wells by rhodamine fluorescence. The enzyme yPIP was then introduced onto the loaded chip and incubated at 37 °C for 2 h. N-terminal amino acid cleavage was assessed by acquiring images of the chip following introduction of yPIP and calculating the percent of green dye lost. Figure 17B shows imaging results from an experiment that exhibited 6-7% loading onto the loading stage and 91% signal loss in pre-loaded wells after incubation with yPIP, which is indicative of N-terminal amino acid cleavage. Figure 17C shows representative signal traces from these experiments, demonstrating that an increase in dye signal is detected upon loading of the labeled peptide, as well as a loss of dye signal following exposure to yPIP.

[0319] As a further confirmation of N-terminal amino acid cleavage at the single molecule level, an on-chip FRET assay was developed to evaluate exopeptidase recognition and cleavage activity. Figure 18A generally depicts the FRET peptide conjugate assay (panel A) and the FRET enzyme conjugate assay (panel B). In the FRET peptide conjugate assay (panel A), the immobilized peptide construct contains a FRET donor label attached to the linker and a FRET acceptor label attached to the N-terminus. N-terminal amino acid cleavage is detected by the loss of signal from the FRET acceptor label upon exposure to peptidase. Additionally, this design allows monitoring of peptide conjugate loading throughout the experiment by tracking the release from the FRET donor label.

[0320] In the FRET enzyme conjugate assay (panel B), the immobilized peptide construct comprises a first label of a FRET pair attached to a linker, and the peptidase is labeled with a second label of the FRET pair. N-terminal amino acid cleavage is detected by an increase in fluorescence attributable to a FRET interaction that occurs when the peptidase is in sufficient proximity to the peptide and has sufficient residence time at the N-terminus. In addition, this assay allows the evaluation of processive amino acid cleavage by a processive exopeptidase by detecting the increase in FRET signal over time due to processive cleavage.

[0321] Figure 18A also shows in panel A the FRET peptide constructs that were designed and synthesized for use in the FRET peptide conjugate assays in panel A. As shown, the FRET peptide constructs contained a rhodamine dye (ATTO 647N) attached to the N-terminal aspartic acid residue of a peptide with an N-terminal penultimate proline residue. The peptide was further conjugated to a solubilizing DNA linker attached to a cyanine dye (Cy3B) for FRET and a biotin moiety for surface immobilization.

[0322] In this experiment, the FRET peptide construct was loaded onto a glass chip with an array of sample wells, and the collected light was filtered first with a green filter and then with a red filter. The loading of the FRET peptide construct was detected by measuring the signal passing through both the green and red filters. Terminal amino acid cleavage was detected when the signal was only measurable with the green filter, suggesting that the red dye-conjugated N-terminal amino acid of the FRET peptide construct was cleaved by yPIP. This detection pattern is illustrated in panel C. As shown, if both dyes are detectable before the addition of yPIP and only the green dye is visible after incubation with yPIP, it can be reasonably concluded that such a change in the detection pattern is due to peptide cleavage and not due to overall photobleaching or loss of peptide. Additionally, an increase in fluorescence from the green dye alone would be expected since its emission is no longer absorbed by the red dye.

[0323] Following loading of the FRET peptide construct onto a chip modified by surface passivation with phosphonic acid and silane, yPIP was introduced and images were obtained at several time points. To assess the overall cleavage trend, the ratio (green) / (green+red) was calculated for each experiment. This ratio increases with the degree of cleavage that occurs. Figure 18B is a plot of the FRET emission ratio across all apertures at different time points incubated with yPIP. As shown, the contribution of the green dye to the fluorescence emission ratio increases over time, suggesting that more N-terminal aspartic acid residues are cut off when incubated with yPIP, leaving a truncated peptide with only the green dye.

[0324] Cutting efficiency was then assessed at different time points by determining at which time points dye fluorescence was observed. This was done by simple thresholding. For example, dye was determined to be present if the average dye emission signal was >2.5 upon excitation (when each corresponding filter was applied). An aperture exhibiting cutting would then exhibit both green and red dye during the loading phase of the experiment, but only green dye at the time point exposed to yPIP. As shown in Figure 18C, progressively more cutting was observed as the chip was exposed to longer incubation times with yPIP. Example signal traces showing cutting at each of the three yPIP treatment time points are shown in Figure 18D.

[0325] Additional experiments were performed with yPIP and other peptidases using chips modified by surface passivation with dextran, yielding similar results showing increased terminal amino acid cleavage over time following the introduction of peptidase onto the chip. Figure 18E is a plot of the FRET emission ratio across the loaded aperture at different time points incubated with yPIP. Figure 18F is a plot of the FRET emission ratio across the loaded aperture at different time points incubated with aminopeptidase. Overall, the experiments herein demonstrate that it is possible to detect N-terminal amino acid cleavage in real time at the single molecule level using different exopeptidases and different labeling strategies.

[0326] Example 5. Terminal amino acid discrimination by labeled affinity reagents To detect N-terminal aromatic residues, we identified adaptor proteins involved in protein degradation pathways as potential candidates for use as labeling affinity reagents. The adaptor protein ClpS2 from α-proteobacteria (A. tumefaciens) was expressed and labeled at exposed cysteine ​​residues. Figure 19A shows the crystal structure of the ClpS2 protein with exposed cysteine ​​residues shown as sticks. The exposed cysteine ​​residues were labeled with a rhodamine dye (ATTO 532).

[0327] To test whether labeled ClpS2 allows N-terminal amino acid discrimination at the single molecule level, peptides with different N-terminal aromatic residues were prepared. Example single molecule intensity traces from these experiments are shown in Figure 19B. As shown, the signal traces demonstrate residue-specific on-off binding patterns corresponding to the labeled affinity reagent reversibly binding to the N-termini of peptides with either an N-terminal phenylalanine residue (F, top signal trace), an N-terminal tyrosine residue (Y, middle signal trace), or an N-terminal tryptophan residue (W, bottom signal trace).

[0328] Further analysis of the single molecule trajectories was performed using the results shown in Figures 19C-19E. Figure 19C is a plot showing the discriminant pulse durations (duration of signal peaks) between the three N-terminal residues when reversibly bound by labeled ClpS2. Figure 19D is a plot showing the discriminant inter-pulse durations (duration between signal pulses) between the three N-terminal residues. Figure 19E shows a plot further illustrating the discriminant pulse durations between phenylalanine, tyrosine, and tryptophan at the peptide N-terminus. The average pulse durations of the different N-terminal residues are visualized by histograms (A)-(B) and layered histograms (C).

[0329] Another adaptor protein, ClpS from Thermosynochoccus elongatus, was evaluated for use as a labeled affinity reagent for leucine recognition. Data from residence time analysis shown in Figures 19F-19H demonstrated that the labeled teClpS protein produced detectable binding interactions with terminal leucine residues of polypeptides with an average pulse duration of 0.71 seconds. The amino acid sequence of the teClpS protein used in these experiments is shown in Table 1.

[0330] Similar experiments were performed to evaluate A. tumefaciens ClpS1 and S. elongatus ClpS2 as potential reagents for leucine recognition and GID4 as a potential reagent for proline recognition. Figure 19I shows example results from retention time analysis demonstrating distinguishable recognition of phenylalanine, leucine, tryptophan, and tyrosine by A. tumefaciens ClpS1. Figure 19J shows example results from retention time analysis demonstrating leucine recognition by S. elongatus ClpS2. Figures 19K-19L show example results from retention time analysis demonstrating proline recognition by GID4.

[0331] Example 6. Polypeptide sequencing by recognition upon digestion Experiments were performed to evaluate peptide sequencing by N-terminal amino acid recognition during ongoing degradation reactions. Example results from these experiments are shown in Figures 20A-20D, which show single molecule intensity traces obtained from two independent polypeptide sequencing reactions performed in real time using labeled ClpS2 protein and aminopeptidase in the same reaction mixture. In each reaction, a polypeptide of sequence YAAWAAFADDDWK (SEQ ID NO: 78) was immobilized to the chip surface via a C-terminal lysine residue by loading the peptide composition (10 pM) onto the chip for 20 min, and the immobilized peptide was monitored in the presence of labeled affinity reagent (500 nM ATTO 542-labeled A. tumefaciens ClpS2-V1) and aminopeptidase cleavage reagent (8 μM VPr).

[0332] Figures 20A and 20C show signal trace data from two different sequencing runs, with the top panels (panel 1 in Figure 20A, panel 2 in Figure 20C) showing full traces, and the bottom panels (Y, W, F) showing magnified regions corresponding to each of the highlighted regions in the full traces. Figures 20B and 20D show histograms of pulse duration statistics for the trace data of the corresponding panels marked in Figures 20A and 20C, respectively. As shown in the full signal traces of each sequencing run (panels 1, 2), three distinct time intervals of signal pulses were observed throughout the course of the reaction. As highlighted by the magnified regions (panels Y, W, F), the three intervals are visually distinguishable from one another based on observable differences in the patterns of signal pulses.

[0333] To further analyze the signal pulse data, pulse duration statistics were determined for each time interval (Figures 20B and 20D). The difference in pulse duration distribution was determined to correspond to that observed for these amino acids individually in steady-state on-chip binding assays with ClpS2, and the signal pulse information was phenotypically consistent between intervals from the sequencing run and the individual amino acid binding assays.

[0334] As confirmed by analysis of the signal pulse information, the three time intervals of signal pulses observed in the progression of each sequencing run correspond to the recognition patterns of Y, W, and F, respectively (panels 1, 2). The intervening time periods between the signal pulse patterns are due to the selectivity of ClpS2-V1 not binding to N-terminal alanine residues. As illustrated by the full signal traces, the first interval corresponds to Y recognition, followed by a pause as VPr peptidase cuts Y and two alanine residues, followed by a second interval corresponding to W recognition, followed by another pause as VPr peptidase cuts W and two alanine residues, and finally a third interval corresponding to F recognition, after which VPr peptidase cuts and removes F, terminating with the remaining ADDDWK peptide. These results indicate that pulse duration information obtained by terminal amino acid recognition during ongoing degradation reactions can be used to determine characteristic patterns that distinguish different types of terminal amino acids.

[0335] Example 7. Identification and cleavage of terminal amino acids with labeled exopeptidase Studies were performed to investigate the feasibility of a single reagent capable of both identifying the terminal amino acid of a peptide and cleaving it from the peptide. As a single reagent, the exopeptidase must be able to bind to the peptide while retaining cleavage activity towards the terminal residue. Therefore, an initial approach was undertaken that utilizes traditional labelling strategies by targeting the natural surface-exposed amino acids of different exopeptidases. In these experiments, surface-exposed cysteines (-SH) or lysines (-NH) were labelled with fluorescent dyes, which have proven to be a robust method for exopeptidase labelling. 2 ) residues were labeled. However, in certain cases, this approach produced a heterogeneous population of proteins that were labeled with more than one dye.

[0336] To gain more precise control over where on the exopeptidase the labeling occurs, ensuring that each exopeptidase molecule is labeled with a single fluorescent dye (and thus eliminating off-target reactivity of the dyes), a new labeling strategy was explored. In these experiments, labeled exopeptidases were prepared using a site-specific labeling strategy in which a non-natural amino acid containing a reactive functional group was introduced into the exopeptidase (see, for example, Chin, JW et al., J Am Chem Soc. 124(31), 7 Aug. 2002, 9026-9027).

[0337] Proline iminopeptidase from Yersinia pestis (yPIP) was modified by mutating the lysine residue at position 287 to a residue bearing a p-azidophenylalanine (pAzF) side chain. Figure 21A shows the crystal structure of yPIP with the mutation suggested by the chemical structure of pAzF shown with the K287 side chain shown as sticks. This mutation site was chosen based on the stability provided by the α-helix at this position and to ensure that the new azide functionality was solvent exposed.

[0338] A pEVOL plasmid was obtained containing a mutant amino-tRNA synthetase and mutant tRNA required to incorporate pAzF into the amino acid chain. An amber stop codon (TAG) required for specific incorporation of pAzF was then introduced into the cDNA using the QuickChange II mutagenesis kit. The cDNA was then sequenced to confirm the TAG codon position. This was followed by co-transfection of both the pET21b+ plasmid containing the yPIP amber mutant and the pEVOL plasmid containing the cellular machinery where the tRNA feeds pAzF to the amber codon. The co-transfected cells were then grown to 0.8 ODU and induced with 0.02% arabinose and 1 mM IPTG in the presence of 2 mM pAzF in 2 L of LB and harvested using chemical lysis. Purification was performed using a 5 mL affinity chromatography column and the protein was eluted with 100 mM imidazole. The resulting protein was then concentrated by dialysis against 50 mM HEPES pH 8.0 and 0.5 M KCl, aliquoted, flash frozen and then stored at -20°C.

[0339] To confirm the presence of azide groups in the purified protein, DBCO-Cy3 (2 mM) was reacted with the pAzF-yPIP variant (220 μM) (reaction conditions: 50 mM HEPES pH 8.0, 0.5 mM KCl, and 20% DMSO, 10 h at 37 °C and 48 h at room temperature). The protein reaction product was purified by size-exclusion chromatography and the resulting protein was judged to be 100% labeled with the azide-reactive DBCO-Cy3 reagent (Figure 21B), indicating robust incorporation of unnatural amino acids.

[0340] SDS-PAGE analysis of unlabeled and labeled pAzF variants confirmed protein labeling and purity of the final product. Figure 21C shows a photograph of an SDS-PAGE gel confirming Cy3 labeling of pAzF-yPIP (an overexposed image of the gel is shown in Figure 21D to show the ladder). Figure 21E shows a photograph of a Coomassie stained gel confirming that both the dye and protein co-migrate and are pure.

[0341] To confirm that the enzyme was still active after labeling and purification, dye-labeled pAzF-yPIP variants were used in activity assays. As shown in Figure 21F, Cy3-pAzF-yPIP was able to hydrolyze 100% of the peptide substrate in 1 h using a 1000-fold excess of substrate as measured by HPLC. These experiments demonstrate a method that allows for site-specific modification and labeling of exopeptidases with minimal perturbation of native protein structure / function.

[0342] Example 8. Recognition of modified amino acids in polypeptide sequencing Experiments were performed to evaluate the recognition of amino acids containing specific post-translational modifications. A triple mutant (T8V, S10A, K15L) of the Src homology 2 (SH2) domain of Fyn tyrosine kinase was tested by peptide sequencing as a potential recognition molecule for phosphorylated tyrosine residues. The mutant protein was immobilized at the bottom of a sample well, a fluorescently labeled peptide containing an N-terminal phosphotyrosine was added, and single molecule signal traces were collected. During these experiments, peptide binding by the immobilized protein was detected, as shown by the representative traces in Figure 22A. Pulse duration data collected during these experiments are shown in Figure 22B (top, middle, and bottom plots correspond to the top, middle, and bottom traces in Figure 22A, respectively). Pulse duration and inter-pulse duration statistics are shown in Figure 22C (top and bottom panels, respectively).

[0343] To confirm that Fyn protein is specific for phosphorylated tyrosine, a control experiment was performed. The experiment was repeated for each of three different peptides: the first peptide containing an N-terminal unmodified tyrosine (Y, FIG. 22D), the second peptide containing an N-terminal penultimate unmodified tyrosine (YY, FIG. 22E), and the third peptide containing an N-terminal phosphoserine (FIG. 22F). As shown, no binding was detected with any of the peptides used in the negative control experiment.

[0344] Example 9. Recognition of penultimate amino acids in polypeptide sequencing Experiments were performed to determine the effect of penultimate amino acids on the pulse duration of A. tumefaciens ClpS2-V1. Forty-nine different fluorescently labeled peptides were prepared that contained a unique dipeptide sequence at the N-terminus, where the N-terminal amino acid was F, W, or Y, and the penultimate position was one of the 20 natural amino acids. In each experiment, ClpS2-V1 was immobilized at the bottom of a sample well, one of the fluorescently labeled peptides was added, and single molecule signal traces were collected for 10–20 min. Pulse duration data were collected in a minimum of 50 sample wells for each peptide.

[0345] FIG. 23 shows the median pulse duration for each of the 50 peptides, with data points grouped by penultimate amino acid (x-axis) and N-terminal amino acid represented with different symbols.

[0346] Example 10. Simultaneous amino acid recognition by multiple recognition molecules To demonstrate terminal amino acid recognition of immobilized peptides by more than one labeled recognition molecule, a single molecule peptide recognition experiment was performed. A single peptide molecule (FYPLPWPDDDY (SEQ ID NO: 79)) containing an N-terminal phenylalanine was immobilized in the sample well of the chip. A buffer solution containing 500 nM each of atClpS1 (Agrobacterium tumifaciens ClpS1, sequence provided in Table 1) and atClpS2-V1 (Agrobacterium tumifaciens ClpS2 variant 1, sequence provided in Table 1) was added, except that atClpS1 and atClpS2-V1 were labeled with Cy3 and Cy3B, respectively. Because the intensity of Cy3B is higher than Cy3, atClpS2-V1 binding events were easily distinguishable from atClpS1 binding events.

[0347] Figures 24A-24C show the results of experiments demonstrating single molecule peptide recognition by differentially labeled recognition molecules. Representative traces are presented in Figure 24A. The pulse duration distributions were distinct for each binder (Figure 24B) and corresponded to their kinetic profiles observed in single binder experiments. The average pulse durations were 1.3 s for atClpS1 and 1.0 s for atClpS2-V1 (Figure 24C). The pulse rates were also distinct, 8.1 pulses / min for atClpS1 and 14.1 pulses / min for atClp2-V1 (Figure 24C). Thus, when more than one recognition molecule is included for dynamic recognition of an immobilized peptide, the binding characteristics of each recognition molecule (including pulse duration, inter-pulse duration, and pulse rate) can simultaneously provide information about the peptide sequence.

[0348] Example 11. Enhancement of photostability by recognition molecule linkers Experiments were performed to assess the photostability of immobilized peptides during single molecule sequencing. Dye-labeled atClpS2-V1, as described in Example 5, was added to sample wells containing immobilized peptide substrate in the presence of 532 nm excitation light to monitor recognition by release from ATTO532. A representative trace is shown in Figure 25A. As shown in the top panel, recognition was observed to cease approximately 600 seconds into the experiment. The bottom panel is a zoomed-in view showing the signal pulse from approximately 180 to 430 seconds into the reaction.

[0349] Figure 25B shows a visualization of the crystal structure of the ClpS2 protein used in these experiments. As shown, the cysteine ​​residue that serves as the dye conjugation site is approximately 2 nm from the terminal amino acid binding site. We hypothesized that photodamage to the peptide is caused by the proximity of the dye to the N-terminus of the peptide upon binding. To mitigate the potential photodamaging effect of dye proximity, the ClpS2 protein was dye-labeled via a linker that increased the distance between the dye and the N-terminus of the peptide to more than 10 nm. The linker included streptavidin and a double-stranded nucleic acid. The double-stranded nucleic acid was labeled with two Cy3B dye molecules and attached to streptavidin via the bis-biotin moiety, and the ClpS2 protein was attached to each of the two remaining binding sites on the streptavidin via the biotin moiety. A representative trace with this dye-shielded ClpS2 molecule is shown in Figure 25C. As shown in the top panel, the recognition time was extended to approximately 6,000 seconds from the beginning of the experiment. The bottom panel is an enlarged view showing the signal pulse from approximately 750 to 930 seconds after the start of the reaction.

[0350] A DNA-streptavidin recognition molecule was generated using a linker containing a double-stranded nucleic acid labeled with two Cy3B dye molecules and attached to streptavidin via a bis-biotin moiety, and a single ClpS2 protein was attached to the two remaining binding sites on streptavidin via the bis-biotin moiety. This construct was used in single-molecule peptide sequencing reactions, and representative traces of these experiments are shown in Figures 26A-26D.

[0351] The sequencing experiment described in Example 6 was repeated with the following change in reaction conditions: a DNA-streptavidin ClpS2 recognition molecule was used in combination with the hTET amino acid cleavage reagent. Representative signal traces are shown in FIG.

[0352] Example 12. Sequencing by recognition during digestion with multiple exopeptidases An experiment was performed to evaluate the use of multiple types of exopeptidases with differential cleavage specificities in single molecule peptide sequencing reaction mixtures. A single peptide molecule (YAAWAAFADDDWK (SEQ ID NO: 78)) was immobilized via the C-terminal lysine residue in the sample well of the chip. A buffer containing atClpS2-V1 for amino acid recognition and hTET for amino acid cleavage was added. A representative trace is presented in Figure 28A, and a zoomed-in view of the pulse pattern region is shown in Figure 28B.

[0353] An experiment was performed to evaluate the sequencing reaction in the presence of two types of exopeptidases with differential specificity. A single peptide molecule (FYPLPWPDDDYK (SEQ ID NO: 80)) was immobilized via the C-terminal lysine residue in the sample well of the chip. A buffer containing atClpS2-V1 for amino acid recognition and both yPIP and hTET for amino acid cleavage was added. A representative trace is presented in Figure 28C, and a zoomed-in view of the pulse pattern region is shown in Figure 28D. Additional representative traces of these reaction conditions are shown in Figure 28E.

[0354] Further experiments were performed to evaluate the sequencing reaction in the presence of two types of exopeptidases with differential specificities. A single peptide molecule (YPLPWPDDDYK (SEQ ID NO: 81)) was immobilized via a C-terminal lysine residue in the sample well of the chip. In one experiment, a buffer containing atClpS2-V1 for amino acid recognition and both yPIP and hTET for amino acid cleavage was added. A representative trace is presented in FIG. 28F, and a zoomed-in view of the pulse pattern area is shown in FIG. 28G. Additional representative traces of these reaction conditions are shown in FIG. 28H. In a further experiment, a buffer (50 mM MOPS, 60 mM KOAc, 200 μM Co(OAc)) containing atClpS2-V1 for amino acid recognition and both PfuTET and yPIP for amino acid cleavage was added. 2 ) was added. A representative trace is presented in Figure 28I, and a zoomed-in view of the pulse pattern region is shown in Figure 28J.

[0355] Equivalents and Scope In the claims, articles such as "a," "an," and "the" may mean one or more, unless indicated to the contrary or otherwise evident from the context. A claim or description including "or" between one or more members of a group shall be deemed satisfied if one, more than one, or all of the group members are present in, utilized in, or otherwise relevant to a given product or process, unless indicated to the contrary or otherwise evident from the context. The invention includes embodiments in which exactly one member of a group is present in, utilized in, or otherwise relevant to a given product or process. The invention includes embodiments in which two or more, or all of the group members are present in, utilized in, or otherwise relevant to a given product or process.

[0356] Moreover, the present invention encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms are introduced from one or more of the enumerated claims into other claims. For example, any claim that depends on another claim can be amended to include one or more limitations found in any other claim that depends on the same base claim. When elements are presented as lists, e.g., in Markush group format, each subgroup of elements is also disclosed, and any element can be removed from the group. In general, when the invention or aspects of the invention are referred to as including certain elements and / or features, it should be understood that the particular embodiment or aspect of the invention consists or consists essentially of such elements and / or features. For simplicity, those embodiments have not been specifically described in this specification in these exact terms.

[0357] The phrase "and / or" as used herein and in the claims should be understood to mean "either or both" of the elements so conjoined, i.e., elements that are present conjunctively in some cases and disjointly in other cases. Multiple elements listed with "and / or" should be interpreted similarly; that is, "one or more" of the elements are so conjoined. Optionally, other elements may be present other than those specifically identified by the "and / or" phrase, whether or not related to the specifically identified elements. Thus, for example, a reference "A and / or B," when used in conjunction with open-ended language such as "comprising," can mean in one embodiment only A (optionally including elements other than B), in another embodiment only A (optionally including elements other than A), in yet another embodiment both A and B (optionally including other elements), etc.

[0358] As used herein and in the claims, "or" should be understood to have the same meaning as "and / or" as defined above. For example, in the case of separate items in a list, "or" or "and / or" shall be interpreted as inclusive, i.e., including at least one, but also including two or more of the elements or listed elements, and optionally including additional unlisted items. Terms clearly specified to the contrary, such as "only one of" or "exactly one of," or "consisting of" when used in the claims, will imply the inclusion of exactly one of the elements or listed elements. In general, the term "or" as used herein shall only be construed as indicating exclusive alternatives (i.e., "one or the o...

Claims

1. Obtaining data on the degradation process of the polypeptide; analyzing the data to determine portions of the data that correspond to amino acids that are sequentially exposed at the termini of the polypeptide during the degradation process; outputting an amino acid sequence corresponding to said polypeptide; The method includes:

2. The method of claim 1 , wherein the data is indicative of the amino acid identity of the termini of the polypeptide during the degradation process.

3. 3. The method of claim 2, wherein the data is indicative of signals generated by one or more amino acid recognition molecules that bind to different types of terminal amino acids at the termini during the degradation process.

4. The method of claim 1 , wherein the data is indicative of a luminescent signal generated during the degradation process.

5. The method of claim 1 , wherein the data is a measure of an electrical signal generated during the degradation process.

6. The method of claim 1 , wherein analyzing the data further comprises detecting a series of disconnection events and determining a portion of the data between successive disconnection events.

7. The method of claim 1 , wherein analyzing the data further comprises determining an amino acid type for each of the discrete portions.

8. 2. The method of claim 1, wherein each of the discrete portions includes a pulse pattern, and analyzing the data further includes determining a type of amino acid for one or more of the portions based on its respective pulse pattern.

9. 9. The method of claim 8, wherein determining the type of amino acid further comprises identifying an amount of time within a portion when the data is above a threshold and comparing the amount of time and duration for the portion.

10. 9. The method of claim 8, wherein determining the type of amino acid further comprises identifying at least one pulse duration for each of the one or more portions.

11. 9. The method of claim 8, wherein determining the type of amino acid further comprises identifying at least one inter-pulse duration for each of the one or more portions.

12. The method of claim 1 , wherein the amino acid sequence comprises a series of amino acids corresponding to the portion.

13. At least one hardware processor; and at least one non-transitory computer-readable storage medium storing processor-executable instructions that, when executed by said at least one hardware processor, cause said at least one hardware processor to perform a method according to any one of claims 1 to 12.

14. At least one non-transitory computer readable storage medium storing processor executable instructions that, when executed by at least one hardware processor, cause the at least one hardware processor to perform the method of any one of claims 1 to 12.

15. 1. A method for polypeptide sequencing, the method comprising: contacting a single polypeptide molecule with one or more terminal amino acid recognition molecules; sequencing the single polypeptide molecule by detecting a series of signal pulses indicative of association of the one or more terminal amino acid recognition molecules with successive amino acids exposed at the termini of the single polypeptide molecule during degradation of the single polypeptide; The method includes:

16. 16. The method of claim 15, wherein association of the one or more terminal amino acid recognition molecules with each type of amino acid exposed at the terminus produces a characteristic pattern of the series of signal pulses that is distinct from other types of amino acids exposed at the terminus.

17. The method of claim 16 , wherein the characteristic pattern comprises a portion of the series of signal pulses.

18. 18. The method of claim 17, wherein the characteristic pattern of signal pulses corresponds to individual association events between a terminal amino acid recognition molecule and an amino acid exposed at the terminus.

19. 20. The method of claim 18, wherein the signal pulses of the characteristic pattern include a pulse duration characteristic of a bond dissociation rate between the terminal amino acid recognition molecule and the amino acid exposed at the terminus.

20. 20. The method of claim 19, wherein each signal pulse of the characteristic pattern is separated from the others by an inter-pulse duration characteristic of the association rate of terminal amino acid recognition molecule binding.

21. 21. The method of any one of claims 16 to 20, wherein the characteristic pattern corresponds to a series of reversible terminal amino acid recognition molecule binding interactions with the amino acids exposed at the termini of the single polypeptide molecule.

22. 22. The method of claim 21, wherein the series of reversible terminal amino acid recognition molecule binding interactions comprises the reversible formation of one binary complex species at the termini of the single polypeptide molecule.

23. 22. The method of claim 21, wherein the series of reversible terminal amino acid recognition molecule binding interactions comprises the reversible formation of different binary complex species at the termini of the single polypeptide molecule.

24. 24. The method of any one of claims 16 to 23, wherein said characteristic pattern is indicative of said amino acids exposed at the termini and amino acids at consecutive positions of said single polypeptide molecule.

25. 25. The method of claim 24, wherein the amino acids exposed at the termini and the amino acids at the consecutive positions are of different types.

26. 26. The method of any one of claims 15 to 25, wherein sequencing comprises identifying successive amino acids of each type that are exposed at the termini of the single polypeptide as it is degraded.

27. 26. The method of any one of claims 15 to 25, wherein sequencing comprises identifying a portion of sequential amino acids of all types that are exposed at the termini of the single polypeptide while the single polypeptide is being degraded.

28. 28. The method of any one of claims 15 to 27, wherein sequencing comprises determining the relative positions of successive amino acids exposed at the termini of the single polypeptide while the single polypeptide is degraded.

29. 29. The method of any one of claims 15 to 28, wherein sequencing comprises identifying at least two consecutive amino acids of the single polypeptide molecule.

30. 30. The method of any one of claims 15 to 29, wherein sequencing comprises identifying at least two non-contiguous amino acids of the single polypeptide molecule.

31. 31. The method of any one of claims 15 to 30, wherein sequencing comprises identifying amino acids exposed at said termini as naturally occurring amino acids, unnatural amino acids, or modified variants thereof.

32. 32. The method of any one of claims 15 to 31, wherein sequencing comprises identifying amino acids exposed at the termini as having side chains that are charged, uncharged, polar, non-polar, hydrophobic, aromatic, or combinations thereof.

33. 33. The method of claim 32, wherein the side chains are negatively or positively charged.

34. 34. The method of any one of claims 15 to 33, wherein sequencing comprises identifying the amino acids exposed at the termini as one type selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, selenocysteine, serine, threonine, tryptophan, tyrosine, and valine.

35. 35. The method of any one of claims 15 to 34, wherein sequencing comprises identifying amino acids exposed at said termini as having post-translational modifications.

36. 36. The method of claim 35, wherein the post-translational modification is selected from acetylation, ADP-ribosylation, caspase cleavage, citrullination, formylation, N-linked glycosylation, O-linked glycosylation, hydroxylation, methylation, myristoylation, NEDDylation, nitration, oxidation, palmitoylation, phosphorylation, prenylation, S-nitrosylation, sulfation, SUMOylation and ubiquitination.

37. The method of any one of claims 15 to 36, wherein the series of signal pulses comprises a series of changes in optical signal magnitude over time.

38. 38. The method of claim 37, wherein each signal pulse of the series comprises a plurality of photon emission events.

39. 40. The method of claim 38, wherein the sequencing further comprises determining one or more of luminescence lifetime, luminescence brightness, luminescence intensity, emission wavelength, emission polarization, and luminescence quantum yield.

40. 40. The method of any one of claims 15 to 39, wherein the series of signal pulses comprises a series of changes in the magnitude of an electrical signal over time.

41. 41. The method of claim 40, wherein the series of signal pulses comprises conductivity transitions corresponding to reversible single-molecule binding interactions.

42. 42. The method of any one of claims 15 to 41, wherein each of the one or more terminal amino acid recognition molecules comprises a recognition protein or a recognition nucleic acid.

43. 43. The method of claim 42, wherein the recognition protein is a degradative pathway protein, a peptidase, an antibody, an aminotransferase, a tRNA synthetase, or an SH2 domain-containing protein, or a fragment thereof.

44. 44. The method of claim 43, wherein the degradative pathway protein is an N-end rule pathway protein or a mutant or variant thereof.

45. 45. The method of claim 44, wherein the N-end rule pathway protein is an Arg / N-end rule pathway protein, an Ac / N-end rule pathway protein, or a Pro / N-end rule pathway protein.

46. 46. ​​The method of claim 45, wherein the N-end rule pathway protein is a Gid protein, a UBR box protein or a UBR box domain-containing fragment thereof, a p62 protein or a ZZ domain-containing fragment thereof, or a ClpS protein.

47. 47. The method of claim 46, wherein the Gid protein is Gid4 or Gid10 from S. cerevisiae or a homologous species thereof.

48. 47. The method of claim 46, wherein the UBR box protein is UBR1 or UBR2 from human, S. cerevisiae, or a homologue thereof.

49. 47. The method of claim 46, wherein the p62 protein is p62 from human, rat, or a homologous species thereof.

50. 47. The method of claim 46, wherein the ClpS protein is ClpS1 or ClpS2 from A. tumifaciens, C. crescentus, E. coli, S. elongatus, P. falciparum, T. elongatus, or a homologous species thereof.

51. The method according to any one of claims 42 to 50, wherein the recognition protein is a synthetic or recombinant protein.

52. 43. The method of claim 42, wherein the recognition nucleic acid is a nucleic acid aptamer.

53. The method of any one of claims 15 to 52, wherein each of the one or more terminal amino acid recognition molecules comprises a detectable label.

54. 54. The method of claim 53, wherein the detectable label is a luminescent label or a conductivity label.

55. The method of any one of claims 15 to 54, wherein the single polypeptide molecule is immobilized on a surface.

56. 56. The method of claim 55, wherein the single polypeptide molecule is immobilized on a surface via a terminus distal to the terminus to which the one or more terminal amino acid recognition molecules are associated.

57. 56. The method of claim 55, wherein the single polypeptide molecule is immobilized via the carboxy terminus.

58. 56. The method of claim 55, wherein the single polypeptide molecule is immobilized via its amino terminus.

59. 59. The method of any one of claims 55 to 58, wherein the single polypeptide molecule is immobilized on the surface via a linker.

60. 60. The method of claim 59, wherein the linker comprises a biomolecule, optionally wherein the biomolecule is an oligonucleotide.

61. 61. The method of any one of claims 15 to 60, wherein the single polypeptide molecule is degraded by a cleavage agent that removes one or more amino acids from the ends of the single polypeptide molecule.

62. 62. The method of claim 61, further comprising detecting a signal indicative of association of the cleavage reagent with the terminus.

63. 63. The method of claim 61 or 62, wherein the cleavage reagent comprises a detectable label.

64. 64. The method of claim 63, wherein the detectable label is a luminescent label or a conductivity label.

65. A method according to any one of claims 15 to 64, wherein the train of signal pulses is a train of real-time signal pulses.

66. At least one hardware processor; at least one non-transitory computer readable storage medium storing processor executable instructions that, when executed by said at least one hardware processor, cause said at least one hardware processor to perform a method according to any one of claims 15 to 65; A system including:

67. At least one non-transitory computer readable storage medium storing processor executable instructions that, when executed by at least one hardware processor, cause the at least one hardware processor to perform the method of any one of claims 15 to 65.

68. 1. A method for sequencing a polypeptide, the method comprising: contacting a single polypeptide molecule in a reaction mixture with a composition comprising one or more terminal amino acid recognition molecules and a cleavage reagent; detecting a series of signal pulses indicative of association of the one or more terminal amino acid recognition molecules with a terminus of the single polypeptide molecule in the presence of the cleavage reagent, the series of signal pulses being indicative of a series of amino acids exposed at the terminus over time as a result of terminal amino acid cleavage by the cleavage reagent; The method includes:

69. 69. The method of claim 68, wherein association of the one or more terminal amino acid recognition molecules with each type of amino acid exposed at the terminus produces a characteristic pattern of the series of signal pulses that is distinct from other types of amino acids exposed at the terminus.

70. 70. The method of claim 69, wherein the characteristic pattern comprises a portion of the series of signal pulses.

71. 71. The method of claim 70, wherein the characteristic pattern of signal pulses corresponds to individual association events between a terminal amino acid recognition molecule and an amino acid exposed at the terminus.

72. 72. The method of claim 71, wherein the signal pulses of the characteristic pattern comprise a pulse duration characteristic of the dissociation rate of the bond between the terminal amino acid recognition molecule and the amino acid exposed at the terminus.

73. 73. The method of claim 72, wherein each signal pulse of said characteristic pattern is separated from the others by an inter-pulse duration characteristic of the association rate of terminal amino acid recognition molecule binding.

74. 74. The method of any one of claims 69 to 73, wherein said characteristic pattern corresponds to a series of reversible terminal amino acid recognition molecule binding interactions with said amino acids exposed at the termini of said single polypeptide molecule.

75. 75. The method of claim 74, wherein the series of reversible terminal amino acid recognition molecule binding interactions comprises the reversible formation of one binary complex species at the termini of the single polypeptide molecule.

76. 75. The method of claim 74, wherein said series of reversible terminal amino acid recognition molecule binding interactions comprises the reversible formation of different binary complex species at the termini of said single polypeptide molecule.

77. 77. The method of any one of claims 69 to 76, wherein said characteristic pattern is indicative of said amino acids exposed at the termini and amino acids at consecutive positions of said single polypeptide molecule.

78. 78. The method of claim 77, wherein the amino acids exposed at the termini and the amino acids at the consecutive positions are of different types.

79. 79. The method of any one of claims 68-78, wherein sequencing comprises identifying successive amino acids of each type that are exposed at the termini of the single polypeptide as the single polypeptide is degraded.

80. 79. The method of any one of claims 68-78, wherein sequencing comprises identifying a portion of sequential amino acids of all types that are exposed at the termini of the single polypeptide while the single polypeptide is being degraded.

81. 81. The method of any one of claims 68-80, wherein sequencing comprises determining the relative positions of successive amino acids exposed at the termini of the single polypeptide while the single polypeptide is degraded.

82. 82. The method of any one of claims 68-81, wherein sequencing comprises identifying at least two consecutive amino acids of the single polypeptide molecule.

83. 83. The method of any one of claims 68-82, wherein sequencing comprises identifying at least two non-contiguous amino acids of the single polypeptide molecule.

84. 84. The method of any one of claims 68 to 83, wherein sequencing comprises identifying amino acids exposed at said termini as naturally occurring amino acids, unnatural amino acids, or modified variants thereof.

85. 85. The method of any one of claims 68-84, wherein sequencing comprises identifying amino acids exposed at the termini as having side chains that are charged, uncharged, polar, non-polar, hydrophobic, aromatic, or combinations thereof.

86. 86. The method of claim 85, wherein the side chain is negatively or positively charged.

87. 87. The method of any one of claims 68-86, wherein sequencing comprises identifying amino acids exposed at the termini as one type selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, selenocysteine, serine, threonine, tryptophan, tyrosine, and valine.

88. 88. The method of any one of claims 68 to 87, wherein sequencing comprises identifying amino acids exposed at said termini as having a post-translational modification.

89. 89. The method of claim 88, wherein the post-translational modification is selected from acetylation, ADP-ribosylation, caspase cleavage, citrullination, formylation, N-linked glycosylation, O-linked glycosylation, hydroxylation, methylation, myristoylation, NEDDylation, nitration, oxidation, palmitoylation, phosphorylation, prenylation, S-nitrosylation, sulfation, SUMOylation and ubiquitination.

90. 90. The method of any one of claims 68 to 89, wherein the series of signal pulses comprises a series of changes in optical signal magnitude over time.

91. 91. The method of claim 90, wherein each signal pulse of the series comprises a plurality of photon emission events.

92. 92. The method of claim 91, wherein the sequencing further comprises determining one or more of luminescence lifetime, luminescence brightness, luminescence intensity, luminescence wavelength, luminescence polarization, and luminescence quantum yield.

93. 93. The method of any one of claims 68 to 92, wherein the series of signal pulses comprises a series of changes in magnitude of an electrical signal over time.

94. 94. The method of claim 93, wherein the series of signal pulses comprises conductivity transitions corresponding to reversible single-molecule binding interactions.

95. 95. The method of any one of claims 68 to 94, wherein each of the one or more terminal amino acid recognition molecules comprises a recognition protein or a recognition nucleic acid.

96. 96. The method of claim 95, wherein the recognition protein is a degradative pathway protein, a peptidase, an antibody, an aminotransferase, a tRNA synthetase, or an SH2 domain-containing protein, or a fragment thereof.

97. 97. The method of claim 96, wherein the degradative pathway protein is an N-end rule pathway protein or a mutant or variant thereof.

98. 98. The method of claim 97, wherein the N-end rule pathway protein is an Arg / N-end rule pathway protein, an Ac / N-end rule pathway protein, or a Pro / N-end rule pathway protein.

99. 99. The method of claim 98, wherein the N-end rule pathway protein is a Gid protein, a UBR box protein or a UBR box domain-containing fragment thereof, a p62 protein or a ZZ domain-containing fragment thereof, or a ClpS protein.

100. 100. The method of claim 99, wherein the Gid protein is Gid4 or Gid10 from S. cerevisiae or a homologous species thereof.

101. 100. The method of claim 99, wherein the UBR box protein is UBR1 or UBR2 from human, S. cerevisiae, or a homologue thereof.

102. 100. The method of claim 99, wherein the p62 protein is p62 from human, rat, or a homologous species thereof.

103. 100. The method of claim 99, wherein the ClpS protein is ClpS1 or ClpS2 from A. tumifaciens, C. crescentus, E. coli, S. elongatus, P. falciparum, T. elongatus, or a homologous species thereof.

104. The method of any one of claims 95 to 103, wherein the recognition protein is a synthetic or recombinant protein.

105. 96. The method of claim 95, wherein the recognition nucleic acid is a nucleic acid aptamer.

106. The method of any one of claims 68 to 105, wherein each of the one or more terminal amino acid recognition molecules comprises a detectable label.

107. 107. The method of claim 106, wherein the detectable label is a luminescent label or a conductivity label.

108. The method of any one of claims 68 to 107, wherein the single polypeptide molecule is immobilized on a surface.

109. 109. The method of claim 108, wherein the single polypeptide molecule is immobilized on the surface via a terminus distal to the terminus to which the one or more terminal amino acid recognition molecules are associated.

110. 109. The method of claim 108, wherein the single polypeptide molecule is immobilized via the carboxy terminus.

111. 109. The method of claim 108, wherein the single polypeptide molecule is immobilized via its amino terminus.

112. The method of any one of claims 108 to 111, wherein the single polypeptide molecule is immobilized on the surface via a linker.

113. 113. The method of claim 112, wherein the linker comprises a biomolecule, optionally wherein the biomolecule is an oligonucleotide.

114. The method of any one of claims 68 to 113, further comprising detecting a signal indicative of association of the cleavage reagent with the terminus.

115. The method of any one of claims 68 to 114, wherein the cleavage reagent comprises a detectable label.

116. 116. The method of claim 115, wherein the detectable label is a luminescent label or a conductivity label.

117. 117. The method of any one of claims 68 to 116, wherein the train of signal pulses is a train of real-time signal pulses.

118. At least one hardware processor; at least one non-transitory computer readable storage medium storing processor executable instructions that, when executed by said at least one hardware processor, cause said at least one hardware processor to perform a method according to any one of claims 68 to 117; A system including:

119. At least one non-transitory computer readable storage medium storing processor executable instructions that, when executed by at least one hardware processor, cause the at least one hardware processor to perform the method of any one of claims 68 to 117.

120. 1. A method for polypeptide sequencing comprising: a) identifying the first amino acid at the terminus of a single polypeptide molecule; b) removing the first amino acid to expose a second amino acid at a terminus of the single polypeptide molecule; c) identifying the second amino acid at the end of the single polypeptide molecule; Including, The method, wherein (a)-(c) are carried out in a single reaction mixture.

121. 121. The method of claim 120, wherein (a) through (c) are performed sequentially.

122. 121. The method of claim 120, wherein (c) is performed before (a) and (b).

123. 123. The method of any one of claims 120-122, wherein the single reaction mixture comprises one or more terminal amino acid recognition molecules.

124. The method of claim 123, wherein the first and second amino acids are identified by detecting a series of signal pulses indicative of association of the one or more terminal amino acid recognition molecules with the termini of the single polypeptide molecule.

125. 125. The method of claim 124, wherein association of the one or more terminal amino acid recognition molecules with the first amino acid produces a characteristic pattern of a series of signal pulses that is different from that of the second amino acid.

126. 126. The method of any one of claims 120 to 125, wherein each of the one or more terminal amino acid recognition molecules comprises a recognition protein or a recognition nucleic acid.

127. 127. The method of claim 126, wherein the recognition protein is a degradative pathway protein, a peptidase, an antibody, an aminotransferase, a tRNA synthetase, or an SH2 domain-containing protein, or a fragment thereof.

128. 128. The method of claim 127, wherein the degradative pathway protein is an N-end rule pathway protein or a mutant or variant thereof.

129. 129. The method of claim 128, wherein the N-end rule pathway protein is an Arg / N-end rule pathway protein, an Ac / N-end rule pathway protein, or a Pro / N-end rule pathway protein.

130. 130. The method of claim 129, wherein the N-end rule pathway protein is a Gid protein, a UBR box protein or a UBR box domain-containing fragment thereof, a p62 protein or a ZZ domain-containing fragment thereof, or a ClpS protein.

131. 131. The method of claim 130, wherein the Gid protein is Gid4 or Gid10 from S. cerevisiae or a homologous species thereof.

132. 131. The method of claim 130, wherein the UBR box protein is UBR1 or UBR2 from human, S. cerevisiae, or a homologue thereof.

133. 131. The method of claim 130, wherein the p62 protein is p62 from human, rat, or a homologous species thereof.

134. 131. The method of claim 130, wherein the ClpS protein is ClpS1 or ClpS2 from A. tumifaciens, C. crescentus, E. coli, S. elongatus, P. falciparum, T. elongatus, or a homologous species thereof.

135. The method of any one of claims 126 to 134, wherein the recognition protein is a synthetic or recombinant protein.

136. The method of claim 126, wherein the recognition nucleic acid is a nucleic acid aptamer.

137. 137. The method of any one of claims 120-136, wherein each of the one or more terminal amino acid recognition molecules comprises a detectable label.

138. 138. The method of claim 137, wherein the detectable label is a luminescent label or a conductivity label.

139. 139. The method of any one of claims 120-138, wherein the single reaction mixture comprises a cleavage reagent that removes one or more amino acids from the ends of the single polypeptide molecules.

140. 140. The method of claim 139, wherein the first amino acid is removed by the cleavage reagent.

141. 141. The method of claim 139 or 140, wherein the cleavage reagent comprises a detectable label.

142. 142. The method of claim 141, wherein the detectable label is a luminescent label or a conductivity label.

143. The method of any one of claims 120 to 142, wherein the single polypeptide molecule is immobilized on a surface.

144. 144. The method of claim 143, wherein the single polypeptide molecule is immobilized on the surface via the end distal to the end where the first amino acid is removed.

145. 144. The method of claim 143, wherein the single polypeptide molecule is immobilized via the carboxy terminus.

146. 144. The method of claim 143, wherein the single polypeptide molecule is immobilized via its amino terminus.

147. 147. The method of any one of claims 143 to 146, wherein the single polypeptide molecule is immobilized on the surface via a linker.

148. 148. The method of claim 147, wherein the linker comprises a biomolecule, optionally wherein the biomolecule is an oligonucleotide.

149. 149. The method of any one of claims 124 to 148, wherein the series of signal pulses is a series of real-time signal pulses.

150. At least one hardware processor; at least one non-transitory computer readable storage medium storing processor executable instructions that, when executed by said at least one hardware processor, cause said at least one hardware processor to perform a method according to any one of claims 120 to 149; A system including:

151. At least one non-transitory computer readable storage medium storing processor executable instructions that, when executed by at least one hardware processor, cause the at least one hardware processor to perform the method of any one of claims 120 to 149.

152. 1. A method for identifying an amino acid of a polypeptide, the method comprising: contacting a single polypeptide molecule with one or more amino acid recognition molecules that bind to said single polypeptide molecule; detecting a series of signal pulses indicative of association of said one or more amino acid recognition molecules with said single polypeptide molecule under polypeptide degradation conditions; identifying a first type of amino acid of the single polypeptide molecule based on a first characteristic pattern of the series of signal pulses; The method includes:

153. 153. The method of claim 152, wherein the first characteristic pattern comprises at least a portion of the series of signal pulses.

154. 154. The method of claim 153, wherein the first characteristic pattern of signal pulses corresponds to individual association events between amino acid recognition molecules and the first type of amino acids.

155. 155. The method of claim 154, wherein the signal pulses of the first characteristic pattern comprise a pulse duration characteristic of the dissociation rate of a bond between the amino acid recognition molecule and the first type of amino acid.

156. 156. The method of claim 155, wherein each signal pulse of the first characteristic pattern is separated from the others by an inter-pulse duration characteristic of the association rate of the amino acid recognition molecule binding.

157. 157. The method of any one of claims 152-156, wherein the first characteristic pattern corresponds to a series of reversible amino acid recognition molecule binding interactions with the first type of amino acids of the single polypeptide molecule.

158. 158. The method of claim 157, wherein the series of reversible amino acid recognition molecule binding interactions comprises the reversible formation of one binary complex species at the terminus of the single polypeptide molecule.

159. 158. The method of claim 157, wherein the series of reversible amino acid recognition molecule binding interactions comprises the reversible formation of distinct binary complex species at the termini of the single polypeptide molecule.

160. 160. The method of any one of claims 152 to 159, wherein said single polypeptide molecule comprises said first type of amino acid at a terminal position of said single polypeptide molecule.

161. 161. The method of claim 160, wherein the first characteristic pattern is indicative of the first type of amino acid at the terminal position and the amino acid type at the consecutive position.

162. 162. The method of claim 161, wherein the amino acids at the terminal position and the consecutive positions are of different types.

163. 163. The method of any one of claims 152-162, wherein said single polypeptide molecule comprises said first type of amino acid at an internal position of said single polypeptide molecule.

164. 164. The method of any one of claims 160-163, wherein the single polypeptide molecule comprises the first type of amino acids at the terminal positions and at non-contiguous internal positions of the single polypeptide molecule.

165. 165. The method of claim 164, wherein the first characteristic pattern is indicative of the first type of amino acid at the terminal positions and amino acid types at non-contiguous internal positions.

166. 166. The method of claim 164 or 165, wherein the first characteristic pattern is indicative of the abundance of the first type of amino acid in the single polypeptide molecule.

167. 167. The method of any one of claims 152-166, wherein identifying comprises identifying said first type of amino acids as naturally occurring amino acids, unnatural amino acids, or modified variants thereof.

168. 168. The method of any one of claims 152-167, wherein identifying comprises identifying said first type of amino acid as having a side chain that is charged, uncharged, polar, non-polar, hydrophobic, aromatic, or a combination thereof.

169. 169. The method of claim 168, wherein the side chain is negatively or positively charged.

170. 170. The method of any one of claims 152-169, wherein identifying comprises identifying the first type of amino acid as one type selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, selenocysteine, serine, threonine, tryptophan, tyrosine, and valine.

171. 171. The method of any one of claims 152-170, wherein identifying comprises identifying said first type of amino acid as having a post-translational modification.

172. 172. The method of claim 171, wherein the post-translational modification is selected from acetylation, ADP-ribosylation, caspase cleavage, citrullination, formylation, N-linked glycosylation, O-linked glycosylation, hydroxylation, methylation, myristoylation, NEDDylation, nitration, oxidation, palmitoylation, phosphorylation, prenylation, S-nitrosylation, sulfation, SUMOylation and ubiquitination.

173. 173. The method of any one of claims 152 to 172, wherein the series of signal pulses comprises a series of changes in optical signal magnitude over time.

174. 174. The method of claim 173, wherein each signal pulse of the series comprises a plurality of photon emission events.

175. 175. The method of claim 174, wherein identifying the first type of amino acid further comprises determining one or more of luminescence lifetime, luminescence brightness, luminescence intensity, luminescence wavelength, luminescence polarization, and luminescence quantum yield.

176. 176. The method of any one of claims 152 to 175, wherein the series of signal pulses comprises a series of changes in electrical signal magnitude over time.

177. 177. The method of claim 176, wherein the first characteristic pattern comprises a conductivity transition corresponding to a reversible single-molecule binding interaction.

178. 178. The method of any one of claims 152 to 177, wherein each of the one or more amino acid recognition molecules comprises a recognition protein or a recognition nucleic acid.

179. 179. The method of claim 178, wherein the recognition protein is a degradative pathway protein, a peptidase, an antibody, an aminotransferase, a tRNA synthetase, or an SH2 domain-containing protein, or a fragment thereof.

180. 180. The method of claim 179, wherein the degradative pathway protein is an N-end rule pathway protein or a mutant or variant thereof.

181. 181. The method of claim 180, wherein the N-end rule pathway protein is an Arg / N-end rule pathway protein, an Ac / N-end rule pathway protein, or a Pro / N-end rule pathway protein.

182. 182. The method of claim 181, wherein the N-end rule pathway protein is a Gid protein, a UBR box protein or a UBR box domain-containing fragment thereof, a p62 protein or a ZZ domain-containing fragment thereof, or a ClpS protein.

183. 183. The method of claim 182, wherein the Gid protein is Gid4 or Gid10 from S. cerevisiae or a homologous species thereof.

184. 183. The method of claim 182, wherein the UBR box protein is UBR1 or UBR2 from human, S. cerevisiae, or a homologue thereof.

185. 183. The method of claim 182, wherein the p62 protein is p62 from human, rat, or a homologous species thereof.

186. 183. The method of claim 182, wherein the ClpS protein is ClpS1 or ClpS2 from A. tumifaciens, C. crescentus, E. coli, S. elongatus, P. falciparum, T. elongatus, or a homologous species thereof.

187. 187. The method of any one of claims 178 to 186, wherein the recognition protein is a synthetic or recombinant protein.

188. The method of claim 178, wherein the recognition nucleic acid is a nucleic acid aptamer.

189. The method of any one of claims 152 to 188, wherein each of the one or more amino acid recognition molecules comprises a detectable label.

190. 190. The method of claim 189, wherein the detectable label is a luminescent label or a conductivity label.

191. 191. The method of any one of claims 152 to 190, wherein the single polypeptide molecule is immobilized on a surface.

192. 192. The method of claim 191, wherein the single polypeptide molecule is immobilized on the surface via one end and the one or more amino acid recognition molecules are attached to the single polypeptide molecule via the other end.

193. 193. The method of claim 192, wherein the single polypeptide molecule is immobilized via the carboxy terminus and the one or more amino acid recognition molecules are attached to the single polypeptide molecule at the amino terminus.

194. 193. The method of claim 192, wherein the single polypeptide molecule is immobilized via the amino terminus and the one or more amino acid recognition molecules are attached to the single polypeptide molecule at the carboxy terminus.

195. 195. The method of any one of claims 191 to 194, wherein the single polypeptide molecule is immobilized on the surface via a linker.

196. 200. The method of claim 195, wherein the linker comprises a biomolecule, optionally wherein the biomolecule is an oligonucleotide.

197. 202. The method of any one of claims 152 to 196, further comprising observing degradation of the single polypeptide molecule.

198. 202. The method of any one of claims 152 to 197, wherein the degradation conditions comprise detecting the series of signal pulses in the presence of a cleavage reagent capable of removing one or more terminal amino acids from an end of the single polypeptide molecule.

199. 200. The method of claim 198, further comprising detecting a signal pulse indicative of association of the cleavage reagent with the single polypeptide molecule.

200. 200. The method of claim 198 or 199, wherein the cleavage reagent comprises a detectable label.

201. 201. The method of claim 200, wherein the detectable label is a luminescent label or a conductivity label.

202. 202. The method of any one of claims 197-201, further comprising identifying a second type of amino acid of said single polypeptide molecule based on a second characteristic pattern of said series of signal pulses.

203. 203. The method of claim 202, wherein the second characteristic pattern of signal pulses is indicative of removal of the first type of amino acid from the single polypeptide molecule.

204. A method according to any one of claims 152 to 203, wherein the series of signal pulses is a series of real-time signal pulses.

205. At least one hardware processor; at least one non-transitory computer readable storage medium storing processor executable instructions that, when executed by said at least one hardware processor, cause said at least one hardware processor to perform a method according to any one of claims 152 to 204; A system including:

206. At least one non-transitory computer-readable storage medium storing processor-executable instructions that, when executed by at least one hardware processor, cause the at least one hardware processor to perform the method of any one of claims 152-204.

207. Formula (I): A-(Y) ｎ -D (I) (In the formula, A is an amino acid binding moiety comprising at least one amino acid recognition molecule; Each Y is a polymer that forms a covalent or non-covalent linking group; n is an integer from 1 to 10, inclusive; and D is a label moiety comprising at least one detectable label, and D is a diameter less than 200 Å. A composition comprising a soluble amino acid recognition molecule of the invention.

208. - (Y) ｎ The composition of claim 207, wherein - is at least 2 nm in length.

209. The composition of claim 207, wherein -(Y)n- is at least 5 nm in length.

210. - (Y) ｎ The composition of claim 207, wherein - is at least 10 nm in length.

211. The composition of any one of claims 207-210, wherein each Y is independently a biomolecule, a polyol, or a dendrimer.

212. The composition of claim 211, wherein the biomolecule is a nucleic acid, a polypeptide, or a polysaccharide.

213. The soluble amino acid recognition molecule has the formula: A - Y １ - (Y) ｍ - D or A - (Y) ｍ - Y １ - D (In the formula, Y １ is a nucleic acid or a polypeptide, and m is an integer between 0 and 10 (inclusive); The composition according to any one of claims 207 to 212, which is one of:

214. 214. The composition of claim 212 or 213, wherein the nucleic acid comprises a first oligonucleotide strand.

215. 215. The composition of claim 214, wherein the nucleic acid comprises a second oligonucleotide strand hybridized to the first oligonucleotide strand.

216. 216. The composition of claim 214 or 215, wherein the nucleic acid forms a covalent linkage via the first oligonucleotide strand.

217. 216. The composition of claim 215, wherein the nucleic acid forms a non-covalent linkage via the hybridized first and second oligonucleotide strands.

218. The composition of any one of claims 212 to 217, wherein the polypeptide is a monovalent or multivalent protein.

219. The composition of claim 218, wherein the monovalent or multivalent protein forms at least one non-covalent linkage via a ligand moiety attached to a ligand binding site of the monovalent or multivalent protein.

220. The composition of claim 219, wherein A, Y, or D comprises a ligand moiety.

221. The soluble amino acid recognition molecule has the formula: A-(Y) ｍ -Y ２ -D or A-Y ２ -(Y) ｍ -D (In the formula, Y ２ is a polyol or a dendrimer, and m is an integer between 0 and 10 (inclusive); The composition according to any one of claims 207 to 212, which is one of:

222. 222. The composition of claim 221, wherein the polyol or dendrimer comprises polyethylene glycol, tetraethylene glycol, poly(amidoamine), poly(propyleneimine), poly(propyleneamine), carbosilane, poly(L-lysine), or one or more combinations thereof.

223. The composition of any one of claims 207-222, wherein A comprises a plurality of amino acid recognition molecules.

224. The composition of claim 223, wherein each amino acid recognition molecule of the plurality is attached to a different attachment site on the Y,.

225. The composition of claim 223, wherein at least two of the multiple amino acid recognition molecules are attached to a single attachment site on the Y,.

226. The composition of any one of claims 207 to 225, wherein the amino acid recognition molecule is a recognition protein or a nucleic acid aptamer.

227. 227. The composition of claim 226, wherein the recognition protein is a degradative pathway protein, a peptidase, an antibody, an aminotransferase, a tRNA synthetase, or an SH2 domain-containing protein, or a fragment thereof.

228. 228. The composition of claim 227, wherein the degradative pathway protein is an N-end rule pathway protein or a mutant or variant thereof.

229. 229. The composition of claim 228, wherein the N-end rule pathway protein is an Arg / N-end rule pathway protein, an Ac / N-end rule pathway protein, or a Pro / N-end rule pathway protein.

230. 230. The composition of claim 229, wherein the N-end rule pathway protein is a Gid protein, a UBR box protein or a UBR box domain-containing fragment thereof, a p62 protein or a ZZ domain-containing fragment thereof, or a ClpS protein.

231. 231. The composition of claim 230, wherein the Gid protein is Gid4 or Gid10 from S. cerevisiae or a homologous species thereof.

232. 231. The composition of claim 230, wherein the UBR box protein is UBR1 or UBR2 from human, S. cerevisiae, or a homologous species thereof.

233. The composition of claim 230, wherein the p62 protein is p62 from human, rat, or a homologous species thereof.

234. The composition of claim 230, wherein the ClpS protein is ClpS1 or ClpS2 from A. tumifaciens, C. crescentus, E. coli, S. elongatus, P. falciparum, T. elongatus, or a homologous species thereof.

235. The composition of any one of claims 207 to 234, wherein the detectable label is a luminescent label or a conductivity label.

236. The composition of claim 235, wherein the luminescent label comprises at least one fluorophore dye molecule.

237. The composition of claim 236, comprising fluorophore dye molecules where D is 20 or less.

238. The composition of claim 236 or 237, wherein the ratio of the number of fluorophore dye molecules to the number of amino acid recognition molecules is from 1:1 to 20:

1.

239. The composition of any one of claims 235 to 238, wherein the luminescent label comprises at least one FRET pair comprising a donor label and an acceptor label.

240. The composition of claim 239, wherein the ratio of the donor label to the acceptor label is 1:1, 2:1, 3:1, 4:1, or 5:

1.

241. The composition of claim 239, wherein the ratio of the acceptor label to the donor label is 1:1, 2:1, 3:1, 4:1, or 5:

1.

242. Formula (II): ___ １ .(). (In the formula, A is an amino acid binding moiety comprising at least one amino acid recognition molecule; Y １ is a nucleic acid or a polypeptide, D is a labeling moiety comprising at least one detectable label; However, Y １ is a nucleic acid, said nucleic acid forms a covalent or non-covalent linking group, and However, Y １ is a polypeptide, the polypeptide is 50×10 －９ Dissociation constant (K Ｄ ) forming a non-covalent linking group characterized by Amino acid recognition molecule.

243. -Y １ The amino acid recognition molecule of claim 242, wherein - is at least 2 nm in length.

244. -Y １ The amino acid recognition molecule of claim 242, wherein - is at least 5 nm in length.

245. -Y １ The amino acid recognition molecule of claim 242, wherein - is at least 10 nm in length.

246. The amino acid recognition molecule of any one of claims 242 to 245, wherein the nucleic acid comprises a first oligonucleotide strand.

247. The amino acid recognition molecule of claim 246, wherein the nucleic acid comprises a second oligonucleotide strand hybridized to the first oligonucleotide strand.

248. The amino acid recognition molecule of claim 247, wherein A is attached to the first oligonucleotide strand and D is attached to the second oligonucleotide strand.

249. 248. The amino acid recognition molecule of claim 246 or 247, wherein A is attached to a first attachment site on the first oligonucleotide strand and D is attached to a second attachment site on the first oligonucleotide strand.

250. 250. The amino acid recognition molecule of any one of claims 246 to 249, wherein each oligonucleotide strand of the nucleic acid comprises less than 150, less than 100, or less than 50 nucleotides.

251. The amino acid recognition molecule of any one of claims 242 to 250, wherein the polypeptide is a monovalent or multivalent protein.

252. The amino acid recognition molecule of claim 251, wherein the monovalent or multivalent protein forms at least one non-covalent linkage via a ligand moiety attached to a ligand binding site of the monovalent or multivalent protein.

253. The amino acid recognition molecule of claim 252, wherein at least one of A and D comprises a ligand portion.

254. The amino acid recognition molecule of any one of claims 242 to 253, wherein the polypeptide is an avidin protein.

255. The amino acid recognition molecule of claim 254, wherein the avidin protein is avidin, streptavidin, traptavidin, tamavidin, bladdervidin, xenavidin, or a homolog or variant thereof.

256. The amino acid recognition molecule of any one of claims 252 to 255, wherein the ligand moiety is a biotin moiety.

257. The above K Ｄ But, 1 x 10 －９ Less than M, 1 x 10 －１０ Less than M, 1 x 10 －１１ Less than M or 1 x 10 －１２ The amino acid recognition molecule of any one of claims 242 to 256, wherein M is less than M.

258. The amino acid recognition molecule has the following formula: A - Y １ - (Y) ｎ - D or A - (Y) ｎ - Y １ - D (In the formula, Each Y is a polymer that forms a covalent or non-covalent linking group; and n is an integer between 1 and 10 (inclusive). The amino acid recognition molecule according to any one of claims 242 to 257, which is one of:

259. The amino acid recognition molecule of claim 258, wherein each Y is independently a biomolecule, a polyol, or a dendrimer.

260. A nucleic acid, at least one amino acid recognition molecule attached to a first attachment site on the nucleic acid; at least one detectable label attached to a second attachment site on the nucleic acid; An amino acid recognition molecule comprising: The amino acid recognition molecule, wherein the nucleic acid forms a covalent or non-covalent linking group between the at least one amino acid recognition molecule and the at least one detectable label.

261. The amino acid recognition molecule of claim 260, wherein the nucleic acid is a double-stranded nucleic acid comprising a first oligonucleotide strand hybridized to a second oligonucleotide strand.

262. The amino acid recognition molecule of claim 261, wherein the first attachment site is on the first oligonucleotide strand and the second attachment site is on the second oligonucleotide strand.

263. The amino acid recognition molecule according to any one of claims 260 to 262, wherein the at least one amino acid recognition molecule is attached to the first attachment site via a protein that forms a covalent or non-covalent linking group between the at least one amino acid recognition molecule and the nucleic acid.

264. 264. The amino acid recognition molecule of any one of claims 260 to 263, wherein the at least one detectable label is attached to the second attachment site via a protein that forms a covalent or non-covalent linking group between the at least one detectable label and the nucleic acid.

265. 265. The amino acid recognition molecule of any one of claims 260 to 264, wherein the first and second attachment sites are separated by 5 to 100 nucleotide bases or nucleotide base pairs on the nucleic acid.

266. a multivalent protein comprising at least two ligand binding sites; at least one amino acid recognition molecule attached to the protein via a first ligand moiety bound to a first ligand binding site on the protein; at least one detectable label attached to the protein via a second ligand moiety bound to a second ligand binding site on the protein; An amino acid recognition molecule comprising:

267. The amino acid recognition molecule of claim 266, wherein the multivalent protein is an avidin protein that contains four ligand binding sites.

268. 268. The amino acid recognition molecule of claim 266 or 267, wherein the ligand binding site is a biotin binding site and the ligand moiety is a biotin moiety.

269. The amino acid recognition molecule of claim 268, wherein at least one of the biotin moieties is a bis-biotin moiety, and the bis-biotin moiety is bound to two biotin binding sites on the avidin protein.

270. 270. The amino acid recognition molecule of any one of claims 266 to 269, wherein the at least one amino acid recognition molecule is attached to the protein via a nucleic acid comprising the first ligand portion.

271. 271. The amino acid recognition molecule of any one of claims 266 to 270, wherein the at least one detectable label is attached to the protein via a nucleic acid that comprises the second ligand moiety.

272. 1. A method for identifying a terminal amino acid of a polypeptide, the method comprising: contacting the polypeptide with one or more labeled affinity reagents that selectively bind to one or more types of terminal amino acids at the termini of the polypeptide; identifying a terminal amino acid at a terminus of said polypeptide by detecting an interaction of said polypeptide with said one or more labeled affinity reagents; The method includes:

273. 1. A method for determining the amino acid sequence of a polypeptide, the method comprising: i. contacting the polypeptide with one or more labeled affinity reagents that selectively bind to one or more types of terminal amino acids at the termini of the polypeptide; ii. identifying a terminal amino acid at a terminus of the polypeptide by detecting an interaction between the polypeptide and the one or more labeled affinity reagents; iii. removing the terminal amino acid; iv. repeating steps (i) to (iv) one or more times at a terminal end of the polypeptide to determine the amino acid sequence of the polypeptide; The method includes:

274. The method further comprising: a. after (i) and before (ii), removing any of the one or more labeled affinity reagents that do not selectively bind to the terminal amino acid; and / or b. after (ii) and before (iii), removing any of the one or more labeled affinity reagents that selectively bind to the terminal amino acid; 274. The method of claim 273, further comprising:

275. (iii) modifying the terminal amino acid by contacting the terminal amino acid with an isothiocyanate; a. contacting the modified terminal amino acid with a protease that specifically binds to and removes the modified terminal amino acid; or b. subjecting the modified terminal amino acid to acidic or basic conditions sufficient to remove the modified terminal amino acid; The method of claim 273, comprising:

276. Identifying the terminal amino acid a. identifying the terminal amino acid as being one type of the one or more types of terminal amino acids to which the one or more labeled affinity reagents bind; or b. identifying the terminal amino acid as being of a type other than the one or more types of terminal amino acid to which the one or more labeled affinity reagents bind; The method of claim 273, comprising:

277. 274. The method of claim 273, wherein the one or more labeled affinity reagents comprise one or more labeled aptamers, one or more labeled peptidases, one or more labeled antibodies, or a combination thereof.

278. The method of claim 277, wherein the one or more labeled peptidases are modified to inactivate cleavage activity or the one or more labeled peptidases retain cleavage activity for removal of (iii).

279. 1. A method for identifying a protein of interest in a mixed sample, the method comprising: cleaving the mixed protein sample to generate a plurality of polypeptide fragments; Determining the amino acid sequence of at least one of the plurality of polypeptide fragments by the method of any one of claims 273 to 278; identifying a protein of interest in the mixed sample if the amino acid sequence is uniquely identifiable to the protein of interest; The method includes:

280. 1. A method for identifying a protein of interest in a mixed sample, the method comprising: cleaving the mixed protein sample to generate a plurality of polypeptide fragments; labeling one or more types of amino acids in said plurality of polypeptide fragments with one or more different light-emitting labels; measuring luminescence over time for at least one of said plurality of labeled polypeptides; determining the amino acid sequence of the at least one labeled polypeptide based on the detected emission; identifying a protein of interest in the mixed sample if the amino acid sequence is uniquely identifiable to the protein of interest; The method includes:

281. 1. A method for identifying the relative positions of two or more amino acids in a polypeptide, the method comprising: labeling two or more amino acids of the polypeptide with one or more types of first FRET labels; detecting a FRET signal from a translocation reaction of the labeled polypeptide in the presence of a cofactor labeled with a second FRET label; determining the relative positions of two or more amino acids based on the FRET signal; The method includes:

282. 1. A method for preparing a protein for analysis, the method comprising: i. blocking free carboxylic acid groups of a protein; ii. denaturing a protein; iii. Blocking free thiol groups of proteins; iv. digesting the protein to produce at least one polypeptide fragment comprising a free C-terminal carboxylic acid group; v. chemically conjugating a functional moiety to said free C-terminal carboxylic acid group; The method includes:

283. 1. A method for preparing a protein for analysis, the method comprising: i. denaturing a protein; ii. Blocking free thiol groups of proteins; iii. digesting the protein to generate at least one polypeptide fragment comprising a free C-terminal carboxylic acid group; iv. blocking the free C-terminal carboxylic acid group to produce at least one polypeptide fragment comprising a blocked C-terminal carboxylic acid group; v. enzymatically conjugating a functional moiety to said blocked C-terminal carboxylic acid group.

Images