Protein sequencing by polymerizable molecule coupling

Abstract

Provided herein are methods and systems for sequencing proteins. One or more methods disclosed herein can use a linker comprising an amino acid reactive group and an additional reactive moiety that can be used to couple a polymerizable molecule. The linker may be coupled to the polymerizable molecule and the amino acid of the peptide and the capture moiety through the polymerizable molecule, followed by cleavage of the amino acid from the peptide. Further processing and analysis may be performed using, for example, nanopores or nanogaps.

Description

[0001] Cross-reference

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 394,475, filed Aug. 2, 2022, which is hereby incorporated by reference in its entirety. BACKGROUND OF THE INVENTION

[0003] Technological improvements in biomolecular analysis and characterization have proven crucial for understanding biological and pathological mechanisms, which are important for disease diagnosis and modeling, the development of therapeutic agents and treatments, and improving health outcomes. Among these technological improvements, nucleic acid sequencing has become an important tool for analyzing the genomes and transcriptomes of biological samples.

[0004] Protein signaling underlies a variety of cellular processes and plays important roles in viruses, cells, and living organisms. However, current techniques for studying proteins are limited in terms of selectivity, sensitivity, throughput, or require prior knowledge. Thus, new methods are needed to characterize and analyze proteins. SUMMARY OF THE INVENTION

[0005] It is recognized herein that there is a need for a technique for studying proteins de novo with greater accuracy and in a high-throughput format. Using nanopores in protein or peptide sequencing by conventional methods presents challenges. The translocation of a peptide or protein through a nanopore and subsequent readout are hindered by the properties of the protein: the protein is folded and unevenly charged, and the close intramolecular spacing of amino acids means that many amino acids enter the nanopore simultaneously, resulting in superimposed signals that are difficult to distinguish from one another. Systems, compositions, kits, and methods for analyzing proteins are provided herein to meet the above needs. The methods of the present disclosure can include attaching multiple polymerizable molecules to the amino acids of a peptide via an intramolecular expansion process and analyzing the polymerizable molecule-amino acid conjugate. One or more of the processes described herein can involve sequencing through a nanopore or nanogap, thereby allowing the identification of individual amino acids of a peptide in the order in which they occur or happen in the peptide.

[0006] In one aspect, provided herein is a method of processing a modified amino acid, comprising: (a) providing the modified amino acid, wherein the modified amino acid comprises a polymerizable molecule; and (b) translocating the modified amino acid or a derivative thereof through a nanopore or nanogap; wherein the polymerizable molecule causes a change in the translocation rate of the modified amino acid or a derivative thereof through the nanopore or nanogap as compared to the translocation rate of an amino acid without the polymerizable molecule.

[0007] In another aspect, the present disclosure provides a method, comprising: (a) providing a modified amino acid, wherein the modified amino acid comprises a polymerizable molecule; and (b) translocating the modified amino acid or a derivative thereof through a nanopore or a nanogap; (c) measuring a signal from the nanopore or the nanogap; wherein the polymerizable molecule results in an increase in the signal-to-noise ratio (SNR) of (c) compared to the measured SNR of an amino acid without the polymerizable molecule.

[0008] In some embodiments, the modified amino acid comprises or is derived from a proteinogenic amino acid conjugated to the polymerizable molecule.

[0009] In some embodiments, the modified amino acid or a derivative thereof further comprises a binder. In some embodiments, the binder comprises an antibody, an antibody fragment, a nanobody, an aptamer, a peptide, a polymer, an inorganic compound, a small molecule, or a derivative thereof.

[0010] In some embodiments, the modified amino acid comprises a non-naturally occurring chemical modification. In some embodiments, the non-naturally occurring chemical modification is a protecting group.

[0011] In some embodiments, the nanopore is conjugated to a helicase. In some embodiments, the helicase guides the modified amino acid or a derivative thereof through the nanopore.

[0012] In some embodiments, (b) is carried out using a topoisomerase or a polymerase.

[0013] In some embodiments, (b) is carried out using an electric field or a magnetic field.

[0014] In some embodiments, the polymerizable molecule comprises a nucleic acid molecule. In some embodiments, the nucleic acid molecule comprises a deoxyribonucleic acid (DNA) molecule, a xeno nucleic acid (XNA) molecule, a ribonucleic acid (RNA) molecule, or a modified variant thereof.

[0015] In some embodiments, the nanopore comprises a transmembrane protein.

[0016] In some embodiments, the nanogap comprises an inorganic material. In some embodiments, the nanogap comprises silicon nitride or molybdenum sulfate.

[0017] In some embodiments, the polymerizable molecule is covalently linked to the modified amino acid.

[0018] In some embodiments, the method further includes generating the modified amino acid from an amino acid of the peptide prior to (a). In some embodiments, the amino acid is located at the terminus of the peptide. In some embodiments, the terminus is the N-terminus. In some embodiments, the modified amino acid includes the polymerizable molecule linked to the terminal amino acid. In some embodiments, the polymerizable molecule is linked to the terminal amino acid through an isothiocyanate moiety.

[0019] In some embodiments, the method further includes, prior to (a), (i) providing a linker, wherein the linker comprises an amino acid-reactive moiety, and (ii) contacting the amino acid-reactive moiety with a protein native amino acid to produce the modified amino acid. In some embodiments, the linker is conjugated to the polymerizable molecule. In some embodiments, the method further includes conjugating the linker to the polymerizable molecule. In some embodiments, the linker comprises a first reactive moiety and the polymerizable molecule comprises a second reactive moiety, and further includes reacting the first reactive moiety with the second reactive moiety to generate the linker conjugated to the polymerizable molecule. In some embodiments, the first reactive moiety or the second reactive moiety comprises a click chemistry moiety. In some embodiments, the polymerizable molecule comprises a nucleic acid linker, wherein the nucleic acid linker comprises a modified nucleobase and a click chemistry moiety. In some embodiments, the amino acid-reactive moiety comprises phenyl isothiocyanate (PITC). In some embodiments, the protein native amino acid is the terminal amino acid of a peptide. In some embodiments, the method further includes tethering the modified amino acid to a capture moiety and cleaving the terminal amino acid from the peptide to produce a remaining peptide and an amino acid-linker-capture moiety (AALC) complex. In some embodiments, the capture moiety is conjugated to a substrate. In some embodiments, the capture moiety comprises a first nucleic acid molecule, and wherein the polymerizable molecule comprises a second nucleic acid molecule. In some embodiments, the first nucleic acid molecule or the second nucleic acid molecule comprises a nucleic acid barcode molecule. In some embodiments, the nucleic acid barcode molecule identifies the peptide. In some embodiments, the nucleic acid barcode molecule contains time information. In some embodiments, the capture moiety comprises a cleavable moiety. In some embodiments, the tethering comprises ligating the first nucleic acid molecule to the second nucleic acid molecule. In some embodiments, the ligation is performed using a ligase. In some embodiments, the ligation includes (i) providing a splint oligonucleotide that comprises a first sequence complementary to at least a portion of the first nucleic acid molecule and a second sequence complementary to at least a portion of the second nucleic acid molecule, and (ii) hybridizing the first sequence to the portion of the first nucleic acid molecule and hybridizing the second sequence to the portion of the second nucleic acid molecule. In some embodiments, the method further includes: (I) providing an additional linker conjugated to an additional nucleic acid molecule, (II) contacting the additional linker with the terminal amino acid of the remaining peptide to produce an additional modified amino acid; and (III) tethering the additional modified amino acid to the AALC complex to produce a derivative thereof.In some embodiments, the method further comprises repeatedly providing another additional linker, contacting the another additional linker with the terminal amino acid of the remaining peptide to produce an additional modified amino acid, and tethering the additional modified amino acid to the AALC complex to produce the derivative thereof, wherein the derivative thereof comprises a plurality of modified amino acids. In some embodiments, the nucleic acid molecule or the additional nucleic acid molecule comprises a barcode sequence. In some embodiments, the nucleic acid molecule comprises a first sequence, wherein the additional nucleic acid molecule comprises a second sequence, wherein the first sequence is different from the second sequence. In some embodiments, the first sequence or the second sequence comprises time or spatial information. In some embodiments, the time information is an index of the number of cycles or iterations of providing the linker or the additional linker.

[0020] In some embodiments, the translocation rate of the modified amino acid is lower compared to the translocation rate of the amino acid without the polymerizable molecule.

[0021] In some embodiments, the method further comprises measuring the current signal of the nanopore or the nanogap to determine the translocation rate.

[0022] In some embodiments, the method further comprises identifying the modified amino acid or its derivative using the nanopore or the nanogap. In some embodiments, the identification comprises measuring the current signal from the nanopore or the nanogap and determining the amino acid type of the modified amino acid or its derivative or its post-translational modification variant.

[0023] In some embodiments, (b) occurs at a temperature lower than the ambient temperature.

[0024] In another aspect, a method of processing a peptide is disclosed herein, comprising: (a) providing the peptide and a linker, wherein the linker is capable of coupling to an amino acid of the peptide; (b) coupling the linker to the amino acid of the peptide; (c) coupling the linker to a capture moiety; (d) cleaving the amino acid from the peptide to produce an amino acid-linker-capture moiety (AALC) complex; (e) providing an additional linker, wherein the additional linker is capable of coupling to another amino acid of the peptide; (f) coupling the additional linker to the another amino acid, thereby producing an additional amino acid-linker complex; and (g) coupling the additional amino acid-linker complex to the AALC complex, thereby producing a stacked AALC complex; wherein the coupling in (c) is performed using a polymerizable molecule.

[0025] In some embodiments, the linker comprises a reactive moiety. In some embodiments, the polymerizable molecule includes an additional reactive moiety capable of reacting with the reactive moiety of the linker, and wherein, prior to (a), the polymerizable molecule is coupled to the linker by reacting the reactive moiety with the additional reactive moiety. In some embodiments, the polymerizable molecule comprises an additional linker, the additional linker comprising the additional reactive moiety, wherein the additional linker comprises a modified nucleobase and the additional reactive moiety comprises a click chemistry moiety.

[0026] In some embodiments, the method further comprises providing the polymerizable molecule prior to (c).

[0027] In some embodiments, the capture moiety is immobilized on a substrate. In some embodiments, the substrate is substantially planar. In some embodiments, the substrate is a bead.

[0028] In some embodiments, the method further comprises providing the capture moiety, wherein the capture moiety comprises a nucleic acid molecule. In some embodiments, the nucleic acid molecule comprises a DNA molecule, an RNA molecule, an XNA molecule, or a modified variant thereof. In some embodiments, the DNA molecule is single-stranded. In some embodiments, the polymerizable molecule includes an additional nucleic acid molecule. In some embodiments, (c) comprises coupling the additional nucleic acid molecule to the nucleic acid molecule of the capture moiety. In some embodiments, the coupling is performed using a ligase. In some embodiments, at least a portion of the additional nucleic acid molecule is complementary to at least a portion of the nucleic acid molecule of the capture moiety. In some embodiments, the coupling is performed by hybridizing at least a portion of the additional nucleic acid molecule with at least a portion of the nucleic acid molecule. In some embodiments, the additional nucleic acid molecule is coupled to the nucleic acid molecule of the capture moiety using a splint oligonucleotide. In some embodiments, the capture moiety comprises a nucleic acid barcode molecule. In some embodiments, the nucleic acid barcode molecule identifies the peptide. In some embodiments, the capture moiety is coupled to the peptide.

[0029] In some embodiments, the amino acid or the other amino acid is an N-terminal amino acid or a C-terminal amino acid.

[0030] In some embodiments, (b) occurs prior to (c).

[0031] In some embodiments, wherein (c) occurs prior to (b).

[0032] In some embodiments, the method further comprises repeating (a)-(g).

[0033] In some embodiments, the method further comprises identifying the AALC complex or the stacked AALC complex. In some embodiments, the identification comprises using a nanopore or a nanogap. In some embodiments, the translocation of the AALC complex or the stacked AALC complex comprises translocating the AALC complex or the stacked AALC complex or a portion thereof through the nanopore or the nanogap. In some embodiments, the translocation occurs at a temperature below ambient temperature. In some embodiments, identifying the AALC complex or the stacked AALC complex comprises determining the amino acid type of the AALC complex or the amino acid type of the stacked AALC complex.

[0034] In some embodiments, the polymerizable molecule comprises a nucleic acid barcode molecule comprising time information.

[0035] In some embodiments, the capture moiety comprises a cleavable moiety.

[0036] In another aspect, provided herein is a method comprising: (a) providing a nucleic acid molecule conjugated to an amino acid or a modified amino acid; (b) sequencing the nucleic acid molecule; and (c) identifying the amino acid or the modified amino acid; wherein (b) and (c) are completed within 1 minute of each other.

[0037] In some embodiments, (b) comprises generating sequencing reads.

[0038] In some embodiments, (b) or (c) is performed using a nanopore. In some embodiments, the nanopore is conjugated to a helicase, a topoisomerase, an unfoldase, or a polymerase.

[0039] In some embodiments, (b) and (c) are performed using a nanopore. In some embodiments, the nucleic acid molecule is conjugated to the amino acid or the modified amino acid via a linker. In some embodiments, prior to (a), the linker comprises an amino acid-reactive moiety and is conjugated to the nucleic acid molecule. In some embodiments, prior to (a), the linker comprises an additional reactive moiety. In some embodiments, the modified amino acid comprises a proteinogenic amino acid, the linker, and the nucleic acid molecule. In some embodiments, the linker comprises at least 2 atoms. In some embodiments, the linker comprises a phenyl isothiocyanate moiety. In some embodiments, the method further comprises, prior to (a), conjugating the linker to the amino acid or the modified amino acid to generate an amino acid-linker complex conjugated to the nucleic acid molecule. In some embodiments, the method further comprises, prior to (b) or (c), conjugating the amino acid-linker complex to a capture moiety, thereby generating an amino acid-linker-capture moiety (AALC) complex. In some embodiments, the capture moiety comprises a nucleic acid molecule. In some embodiments, prior to (a), the amino acid or the modified amino acid is conjugated to a peptide, and further comprises cleaving the AALC complex from the peptide. In some embodiments, the capture moiety comprises a nucleic acid barcode molecule that recognizes the peptide. In some embodiments, the method further comprises providing an additional linker, conjugating the additional linker to an additional amino acid of the peptide to generate an additional amino acid-linker complex, and conjugating the additional amino acid-linker complex to the AALC complex, thereby generating a stacked AALC complex. In some embodiments, the capture moiety comprises a cleavable moiety.

[0040] In some embodiments, the nucleic acid molecule comprises time information.

[0041] In some embodiments, the identification comprises measuring a current signal from the amino acid or the modified amino acid using a nanopore or a nanogap and determining the amino acid type of the amino acid or the modified amino acid or a post-translationally modified variant thereof. In some embodiments, the measurement occurs at a temperature below ambient temperature.

[0042] In some embodiments, the nucleic acid molecule comprises a modified nucleobase and a click chemistry moiety.

[0043] In another aspect, the present disclosure provides a composition comprising: a linker covalently attached to a nucleic acid barcode molecule, wherein the linker comprises an amino acid-reactive group, and wherein the nucleic acid barcode molecule comprises time information.

[0044] In some embodiments, the amino acid reactive group comprises an isothiocyanate. In some embodiments, the isothiocyanate is PITC. In some embodiments, the nucleic acid barcode molecule comprises a modified nucleobase and a click chemistry moiety.

[0045] Another aspect of the present disclosure provides a non - transitory computer - readable medium that includes machine - executable code which, when executed by one or more computer processors, implements any of the methods described above or elsewhere herein.

[0046] Another aspect of the present disclosure provides a system that includes one or more computer processors and computer memory coupled thereto. The computer memory includes machine - executable code which, when executed by one or more computer processors, implements any of the methods described above or elsewhere herein.

[0047] From the following detailed description, additional aspects and advantages of the present disclosure will become apparent to those of ordinary skill in the art, in which only illustrative embodiments of the present disclosure are shown and described. As will be recognized, the present disclosure is capable of other and different embodiments, and several details thereof can be modified in various obvious aspects, all without departing from the present disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature and not restrictive.

[0048] Incorporated by reference

[0049] All publications, patents, and patent applications mentioned in this specification are hereby incorporated by reference in their entirety as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent that the publications and patents or patent applications incorporated by reference conflict with the disclosure contained herein, the specification is intended to supersede and / or take precedence over any such conflicting material. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments in which the principles of the invention are utilized, and to the accompanying drawings (also referred to herein as “FIGURES” and “FIG.”), in which:

[0051] Figure 1 An exemplary workflow of peptide sequencing disclosed herein is schematically shown.

[0052] Figure 2 Another exemplary workflow of peptide sequencing disclosed herein is schematically shown.

[0053] Figure 3Schematically shows another exemplary workflow of peptide sequencing disclosed herein.

[0054] Figure 4 Schematically shows an exemplary composition including a linker and a polymerizable molecule.

[0055] Figure 5 Shows a computer system programmed or otherwise configured to implement the methods provided herein.

[0056] Figure 6A Shows a schematic diagram of an exemplary modified amino acid or its derivative containing a polymerizable molecule and an amino acid-linker complex of three different amino acid types (Asp, Trp, and Tyr). Figure 6B Shows exemplary current traces obtained from a nanopore sequencing system of two control molecules and three amino acid-linker-polymerizable molecule complexes. Figure 6C Shows an exemplary current trace varying with time. Figure 6D Shows an exemplary current trace varying with base pair position. Figure 6E Shows exemplary analysis data for classification of different molecular types.

[0057] Figure 7A Shows a schematic diagram of another exemplary modified amino acid or its derivative containing a polymerizable molecule. Figure 7B Shows an exemplary current trace varying with time. Figure 7C Shows an exemplary current trace varying with base pair position.

[0058] Figure 8A Shows a schematic diagram of another exemplary modified amino acid or its derivative including a polymerizable molecule. Figure 8B Shows the variation of an exemplary current trace with time and base pair position. Figure 8C Shows the variation of an exemplary current trace with time, comparing two polymerizable molecules including different linkers. Figure 8D Shows a t-SNE plot of a dynamic time warping correlation matrix.

[0059] Figure 9 Shows exemplary data on the residence times of two control molecules and three amino acid-linker-polymerizable molecule complexes.

[0060] Figure 10A Shows a schematic diagram of the stacked amino acid-linker-polymerizable molecule complex described herein. Figure 10B Shows a schematic diagram of an exemplary model stacked amino acid-linker-polymerizable molecule complex. Figure 10C Shows a table of exemplary amino acid types included in the model stacked amino acid-linker-polymerizable molecule complex. Figure 10DShows an example current trace as a function of time and base pair position. Figure 10E Shows example classification data for different molecular types.

[0061] Figure 11A Shows an example current trace obtained when a polymerizable molecule translocates through a nanopore sequencing system at different temperatures. Figure 11B Shows a histogram of the translocation duration of molecules through a nanopore at different temperatures. Figure 11C Shows a histogram of the translocation duration of different sequences of a polymerizable molecule at different temperatures. Figure 11D Shows an example current trace of a portion of a polymerizable molecule. Figure 11E Shows an example of the residence time of a portion of a polymerizable molecule under two different temperature conditions. Detailed Description

[0062] Although various embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Many variations, changes, and substitutions will be apparent to those skilled in the art without departing from the present invention. It should be understood that various alternatives to the embodiments of the present invention described herein may be employed.

[0063] Definitions

[0064] When the terms "at least", "greater than", or "greater than or equal to" precede the first value in a series of two or more numerical values, the terms "at least", "greater than", or "greater than or equal to" apply to each value in the numerical series. For example, greater than or equal to 1, 2, or 3 is equivalent to greater than or equal to 1, greater than or equal to 2, or greater than or equal to 3.

[0065] When the terms "not more than", "less than", or "less than or equal to" precede the first value in a series of two or more numerical values, the terms "not more than", "less than", or "less than or equal to" apply to each value in the numerical series. For example, less than or equal to 3, 2, or 1 is equivalent to less than or equal to 3, less than or equal to 2, or less than or equal to 1.

[0066] Reference to "one embodiment", "an embodiment", "example embodiment", "some embodiments", "certain embodiments", "various embodiments", etc., indicates that an embodiment of the disclosed technology so described may include a particular feature, structure, or characteristic, but not every embodiment necessarily includes that particular feature, structure, or characteristic. Moreover, repeated use of the phrase "in one embodiment" does not necessarily refer to the same embodiment, although it may.

[0067] In this document, ranges may be expressed as from “about” or “approximately” or “substantially” a particular value and / or to “about” or “approximately” or “substantially” another particular value. When expressing such a range, other exemplary embodiments include from one particular value and / or to another particular value. Further, the term “about” means within an acceptable error range of a particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measuring system. For example, according to the practice in the art, “about” can mean within an acceptable standard deviation. Alternatively, “about” can mean within a range of up to ±20%, preferably up to ±10%, more preferably up to ±5%, and still more preferably up to ±1% of a given value. Alternatively, especially with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within a factor of 2 of the value. When a particular value is described in this application and the claims, the term “about” is implicit and means within an acceptable error range of that particular value, unless otherwise indicated.

[0068] “Comprising” or “containing” or “including” means that at least the compound, element, particle or method step so designated is present in the composition or article or method, but does not exclude the presence of other compounds, materials, particles, method steps, even if such other compounds, materials, particles, method steps have the same function as those so designated.

[0069] Throughout this specification, various components having particular values or parameters may be identified; however, these items are provided as exemplary embodiments. In fact, the exemplary embodiments do not limit the various aspects and concepts of the disclosure, as many comparable parameters, dimensions, ranges and / or values may be implemented. The terms “first,” “second,” etc., “primary,” “secondary,” etc., do not denote any order, quantity or importance, but rather are used to distinguish one element from another.

[0070] As used herein, the term "protein" generally refers to a molecule comprising two or more amino acids linked by peptide bonds. A protein may also be referred to as a "polypeptide", "oligopeptide", or "peptide". A protein may be a naturally occurring molecule or a synthetic molecule. A protein may comprise one or more non-natural amino acids, modified amino acids, or non-amino acid linkers. A protein may contain D-amino acid enantiomers, L-amino acid enantiomers, or both. The amino acids of a protein may be modified naturally or synthetically, such as by post-translational modification or by chemical modification. In some cases, different proteins may be distinguished from one another based on the different genes they are expressed from in an organism, different primary sequence lengths, or different primary sequence compositions. However, proteins expressed from the same gene may be different proteoforms, e.g., distinguished based on non-identical lengths, non-identical amino acid sequences, or non-identical post-translational modifications. Different proteins may be distinguished based on one or both of the originating gene and the proteoform status.

[0071] As used herein, the term "peptide" may refer to any short single peptide chain. The length of a peptide may be no more than about 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 5, or fewer than about 5 amino acids. A peptide may have a known or unknown biological function or activity. A peptide may include natural, synthetic, modified, or degraded proteins or peptides, or combinations thereof.

[0072] As used herein, the term "single analyte" may refer to an analyte that is manipulated alone or distinguished from other analytes. A single analyte may include biomolecules or synthetic molecules. A single analyte may include small molecules. A single analyte may be a single molecule (e.g., a single biomolecule such as a single protein, nucleic acid molecule, affinity reagent, lipid, carbohydrate, etc.), a single complex of two or more molecules (e.g., a multimeric protein having two or more separable subunits, a single protein attached to a nucleic acid molecule, or a single protein attached to an affinity reagent), a single particle, etc. References herein to "single analyte" in the context of a composition, system, or method do not necessarily exclude the application of the composition, system, or method to multiple single analytes that are manipulated alone or distinguished, unless the context dictates or clearly states otherwise.

[0073] As used herein, "polypeptide" refers to two or more amino acids joined together by peptide bonds. The term "polypeptide" includes proteins having a C-terminus and an N-terminus as commonly known in the art, and may be derived from synthetic or naturally occurring sources. As used herein, "at least a portion of a polypeptide" refers to two or more amino acids in a polypeptide. A polypeptide may comprise one or more peptides. Optionally, a portion of a polypeptide includes at least: 1, 5, 10, 20, 30, or 50 amino acids of the complete amino acid sequence of the polypeptide, whether contiguous or having gaps, or of the full amino acid sequence of the polypeptide.

[0074] As used herein, "immobilization" refers to the attachment between a polypeptide and a substrate such that at least a portion of the polypeptide and the substrate remain in physical proximity. The term "immobilization" includes indirect or direct attachment, which may be reversible or irreversible; for example, the attachment is optionally a covalent or non-covalent bond.

[0075] As used herein, the term "sample" refers to a collected substance or material that contains or is suspected of containing one or more analytes of interest (e.g., biomolecules such as polypeptides). Samples can be modified for purposes such as storage or stability. Samples can be naturally occurring or synthetic. Samples can be processed to separate or remove unwanted fractions or impurities from the analytes of interest. Samples can be enriched or purified. For example, a sample can comprise fractions from separation processes (e.g., chromatography, fractionation, electrophoresis, etc.). Alternatively, a sample can be left unprocessed to separate or remove any unwanted fractions or impurities from the analytes of interest. Samples can be obtained from any suitable source or location, including from organisms, cells, tissues, cell preparations, cell-free compositions, the environment (e.g., air, water, dirt, soil, agriculture, soil, dust). Samples can be obtained from an organism or part of an organism, such as from fluids, tissues, or cells. Samples can include biological and / or non-biological components. As used herein, the term "biological sample" or "biological source" refers to a sample derived from a primarily biological system or organism, such as one or more virus particles, cells (e.g., personalized cells), organelles (e.g., personalized organelles), tissues, body fluids, bone, cartilage, and exoskeleton. A biological sample can comprise a majority of biological material on a mass basis, excluding the weight of fluids within the sample. A biological sample can contain one or more proteins, referred to herein as a protein sample. Biological samples can be obtained from a variety of sources, e.g., from clinical patient samples such as blood, serum, plasma, cerebrospinal fluid (CSF), saliva, mucosal secretions, urine, lymph fluid, sweat, vaginal fluid, semen, etc. Biological samples can be processed to purify and retain one or more biomolecules (e.g., proteins, nucleic acids, carbohydrates, lipids, glycoproteins, lipoproteins, metabolites, etc.) from the biological sample. Biological samples (e.g., protein samples) can be derived from cultured cells, which can be processed or unprocessed. Biological samples (e.g., protein samples) can also be generated from tissue samples such as biopsy samples, which can optionally be processed to release the biomolecules (e.g., proteins) contained therein. Tissue samples can also be derived from in vivo samples, including fresh, frozen, urgent, and fixed tissues.

[0076] As used herein, the terms “antibody” and “immunoglobulin” generally refer to proteins that can recognize and bind to a specific antigen. An antibody or immunoglobulin can refer to an antibody isotype, a fragment of an antibody, including but not limited to Fab, Fv, scFv, and Fd fragments, chimeric antibodies, humanized antibodies, single-chain antibodies, and fusion proteins comprising the antigen-binding portion of an antibody and a non-antibody protein. An antibody can be detectably labeled, e.g., with a fluorophore, a radioisotope, an enzyme (e.g., peroxidase) that generates a detectable product, a fluorescent protein, a nucleic acid barcode sequence, etc. An antibody can also be conjugated to other moieties, such as a member of a specific binding pair, e.g., biotin (a member of the biotin-avidin specific binding pair), etc. The term also includes Fab′, Fv, F(ab′)2, and other antibody fragments that retain specific binding to an antigen. Antibodies can exist in a variety of other forms, including, e.g., Fv, Fab, and (Fab)2, as well as bifunctional (e.g., bispecific) hybrid antibodies (e.g., Lanzavecchia et al., Eur. J. Immunol. 17, 105 (1987)) and single-chain (e.g., Huston et al., Proc. Natl. Acad. Sci. U.S.A., 85, 5879-5883 (1988) and Bird et al., Science, 242, 423-426 (1988), which are incorporated herein by reference). (See generally Hood et al., Immunology, Benjamin, N.Y., 2nd ed. (1984) and Hunkapiller and Hood, Nature, 323, 15-16 (1986), which are incorporated herein by reference).

[0077] As used herein, “binding” generally refers to a covalent or non-covalent interaction between two molecules (referred to herein as “binding partners,” e.g., a substrate and an enzyme or an antibody and an epitope). The binding between binding partners can be specific or non-specific.

[0078] As used herein, “specifically binds” or “binds specifically” generally refers to an interaction between binding partners (e.g., a binding partner and a cognate molecule) such that the binding partner binds to one partner but not to other molecules that may be present in the environment (e.g., in a biological sample, in a tissue, in an in vitro assay) under a set of conditions. A specific binding interaction can involve a binding partner binding to a cognate molecule. Compared to the binding of a binding partner to a non-cognate molecule, a specific binding interaction can involve the binding partner binding to its cognate molecule at a significantly or substantially higher level or with greater affinity. A specific binding interaction can involve a first binding partner that has a greater selectivity for binding to a cognate molecule compared to non-cognate molecules.

[0079] The terms "nucleic acid", "nucleic acid molecule", "oligonucleotide", and "polynucleotide" may be used interchangeably herein and generally refer to polymeric forms of nucleotides or their derivatives, whether occurring naturally or synthetically, of any length. Nucleic acid molecules may comprise one or more deoxynucleotides, deoxynucleoside triphosphates, dideoxynucleoside triphosphates, ribonucleotides, hexitol nucleotides, cyclohexane nucleotides, or analogs or combinations thereof. Nucleic acid molecules may include, for example, DNA, RNA, HNA, CeNA, and modified forms thereof. Nucleic acid molecules may comprise nucleotides linked by phosphodiester bonds. Nucleic acid molecules may have any two-dimensional or three-dimensional structure and may perform any known or unknown function. Nucleic acid molecules may be single-stranded, double-stranded, or partially double-stranded. Non-limiting examples of polynucleotides include genes, gene fragments, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, non-coding DNA, small interfering RNA, short hairpin RNA, microRNA, scaRNA, ribozymes, riboswitches, viral RNA, complementary DNA (cDNA), cosmid DNA, mitochondrial DNA, chromosomal or genomic DNA, viral DNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, control regions, isolated RNA of any sequence, nucleic acid probes, nucleic acid adaptors, and primers. Nucleic acid molecules may be linear, circular, or of any other geometric shape. Examples of polynucleotide analogs include, but are not limited to, xeno nucleic acid (XNA), bridged nucleic acid (BNA), glycol nucleic acid (GNA), hexitol nucleic acid (HNA), cyclohexane nucleic acid (CeNA), peptide nucleic acid (PNA), γPNA, morpholino polynucleotides, locked nucleic acid (LNA), threose nucleic acid (TNA), 2'-O-methyl polynucleotides, 2'-O-alkylribosyl-substituted polynucleotides, phosphorothioate polynucleotides, and boranophosphate polynucleotides. Polynucleotide analogs may have purine or pyrimidine analogs, including, for example, 7-deazapurine analogs, 8-halopurine analogs, 5-halopyrimidine analogs, or universal base analogs that can pair with any base, including inosine, nitroazole, isoquinolone analogs, oxazole carboxamides, and aromatic triazole analogs, or base analogs with additional functionality, such as a biotin moiety for affinity binding.

[0080] As used herein, the term "amino acid" generally refers to an organic compound that combines to form proteins or peptides. Amino acids generally contain an amino group, a carboxylic acid group, and a side chain specific to each amino acid, which serves as the monomeric subunit of a peptide. Amino acids can include 20 standard, naturally occurring, or typical amino acids, as well as non-standard amino acids. Standard, naturally occurring, or typical amino acids include alanine (A or Ala), cysteine (C or Cys), aspartic acid (D or Asp), glutamic acid (E or Glu), phenylalanine (F or Phe), glycine (G or Gly), histidine (H or His), isoleucine (I or Ile), lysine (K or Lys), leucine (L or Leu), methionine (M or Met), asparagine (N or Asn), proline (P or Pro), glutamine (Q or Gln), arginine (R or Arg), serine (S or Ser), threonine (T or Thr), valine (V or Val), tryptophan (W or Trp), and tyrosine (Y or Tyr). Amino acids can be L-amino acids or D-amino acids. Non-standard amino acids can be naturally occurring or chemically synthesized modified amino acids, amino acid analogs, amino acid mimetics, non-standard proteinogenic amino acids, or non-proteinogenic amino acids. Examples of non-standard amino acids include, but are not limited to, selenocysteine, pyrrolysine, and N-formylmethionine, (3-amino acids, homo-amino acids, proline and pyruvate derivatives, 3-substituted alanine derivatives, glycine derivatives, ring-substituted phenylalanine and tyrosine derivatives, linear core amino acids, and N-methyl amino acids.

[0081] As used herein, the term "amino acid type" generally refers to one of the standard, naturally occurring, or canonical amino acids, e.g., alanine (A or Ala), cysteine (C or Cys), aspartic acid (D or Asp), glutamic acid (E or Glu), phenylalanine (F or Phe), glycine (G or Gly), histidine (H or His), isoleucine (I or Ile), lysine (K or Lys), leucine (L or Leu), methionine (M or Met), asparagine (N or Asn), proline (P or Pro), glutamine (Q or Gln), arginine (R or Arg), serine (S or Ser), threonine (T or Thr), valine (V or Val), tryptophan (W or Trp), tyrosine (Y or Tyr), derivatives thereof, and modified forms of any of the foregoing amino acids. The term "amino acid type" can be used herein to distinguish multiple amino acids that contain different side chain groups, as opposed to multiple amino acids that are the same (e.g., amino acids at different positions of a single peptide that have the same side chain). An amino acid type can include a modified version of one of the standard, naturally occurring, or canonical amino acids, e.g., post-translational modifications, epigenetic modifications, or chemical or enzymatic modifications.

[0082] As used herein, the term "post-translational modification" refers to a modification that occurs on a peptide after translation. Post-translational modifications can be covalent modifications or enzymatic modifications. Examples of post-translational modifications include, but are not limited to, acylation, acetylation, alkylation (including methylation), biotinylation, butyrylation, carbamylation, carbonylation, deamidation, deiminiation, diphthamide formation, disulfide bridge formation, eliminylation, flavin attachment, formylation, γ-carboxylation, glutamylation, glycylation, glycosylation, glycosylphosphatidylinositolation, heme C attachment, hydroxylation, hydroxylysine formation, iodination, isoprenylation, lipidation, lipoylation, malonylation, methylation, myristoylation, oxidation, palmitoylation, polyethylene glycolylation, phosphopantetheinylation, phosphorylation, prenylation, propionylation, retinal Schiff base formation, S-glutathionylation, S-nitrosylation, S-sulfinylation, selenylation, succinylation, sulfenylation, ubiquitination, and C-terminal amidation. Post-translational modifications include modifications of the amino terminus and / or carboxy terminus of a peptide. Modifications of the terminal amino group include, but are not limited to, deamination, N-lower alkyl, N-di-lower alkyl, and N-acyl modifications. Modifications of the terminal carboxy group include, but are not limited to, amide, lower alkyl amide, dialkyl amide, and lower alkyl ester modifications (e.g., where the lower alkyl is C 1 -C 4(alkyl). Post-translational modifications also include modifications to amino acids that fall between the amino and carboxyl termini, such as, but not limited to, those described above. The term post-translational modification can also include peptide modifications that include one or more detectable labels. Post-translational modifications can be naturally occurring or synthetic.

[0083] As used herein, the term "binding agent" refers to a molecule that binds, associates, joins, recognizes, or combines with another molecule, such as a nucleic acid molecule, peptide, polypeptide, protein, carbohydrate, synthetic molecule, or small molecule. A binding agent can bind to a macromolecule or a component or feature of a macromolecule. A binding agent can form a covalent or non-covalent association with a molecule, macromolecule, or a component or feature of a macromolecule. A binding agent can also be a chimeric binding agent composed of two or more types of molecules, such as a nucleic acid molecule-peptide chimeric binding agent, a carbohydrate-peptide chimeric binding agent, or a lipid-peptide chimeric binding agent. A binding agent can be a naturally occurring, synthetically produced, or recombinantly expressed molecule. A binding agent can bind to a single monomer or subunit of a macromolecule (e.g., a single amino acid of a peptide) or to multiple linked subunits of a macromolecule (e.g., a dipeptide, tripeptide, or higher-order peptide of a longer peptide, polypeptide, or protein molecule). A binding agent can bind to a linear molecule or a molecule having a three-dimensional structure (also referred to as a conformation). For example, an antibody binding agent can bind to a linear peptide, polypeptide, or protein, or to a conformational peptide, polypeptide, or protein. A binding agent can bind to an N-terminal peptide, C-terminal peptide, or middle peptide of a peptide, polypeptide, or protein molecule. A binding agent can bind to an N-terminal amino acid, C-terminal amino acid, or middle amino acid of a peptide molecule. A binding agent can preferably bind to a chemically modified or labeled amino acid, as compared to an unmodified or unlabeled amino acid. For example, a binding agent can preferably bind to an amino acid that has been modified with an acetyl group, amidino group, dansyl group, PTC group, DNP group, SNP group, etc., as compared to an amino acid that does not have such a group. A binding agent can bind to a post-translational modification of a peptide molecule. A binding agent can exhibit selective binding to a component or feature of a macromolecule (e.g., a binding agent can selectively bind to one of 20 possible natural amino acid residues and bind to or not bind to the other 19 natural amino acid residues with very low affinity). A binding agent can exhibit less selective binding, where the binding agent is capable of binding to multiple components or features of a macromolecule (e.g., a binding agent can bind to two or more different amino acid residues with similar affinity). A binding agent can contain a tag, which can be coupled to the binding agent via a linker.

[0084] As used herein, the term "linker" generally refers to a molecule or moiety involved in joining two or more molecules. A linker can facilitate covalent or non-covalent interactions between two or more molecules. A linker can be a crosslinker. A linker can be mono-functional, bi-functional, tri-functional, tetra-functional, or multi-functional. A linker can be or include nucleotides, nucleotide analogs, amino acids, peptides, polypeptides, or non-nucleotide chemical moieties, such as organic or inorganic compounds. A linker can include polymers, such as polyethylene glycol (PEG), polyethylene, polypropylene, polyvinyl chloride, polystyrene, or other organic or inorganic polymers. A linker can include one or more reactive ends, such as amine-reactive groups, carboxyl-reactive groups, thiol-reactive groups, hydroxyl-reactive groups, etc. Alternatively, a linker can not include reactive ends. In some examples, a linker can be used to join different molecular types, e.g., different biomolecular types (such as peptides and nucleic acid molecules, lipids and peptides, carbohydrates and peptides, etc.); non-biological molecular types; or biomolecules to non-biological molecules. For example, a linker can be used to join a binder to a tag, a tag to a macromolecule (e.g., a peptide, a nucleic acid molecule), a macromolecule to a solid support, a tag to a solid support, etc. A linker can join two molecules via an enzymatic reaction or a chemical reaction (e.g., click chemistry). A linker can join more than two molecules, e.g., via an enzymatic or chemical reaction.

[0085] As used herein, the term "conjugation" generally refers to a covalent or ionic interaction between two entities (e.g., molecules, compounds, or combinations thereof).

[0086] As used herein, the term "tag" generally refers to a molecule or moiety conjugated to a molecule. A tag can include a detectable label, e.g., a fluorophore or fluorescent protein, a radioisotope, an enzyme (e.g., a chromogenic or fluorescent protein, a protein that can catalyze a chromogenic substrate), a mass tag, a hapten (e.g., biotin, digoxin, urushiol, fluorescein), a vibrational or FTIR tag (e.g., an alkyne group). A tag can include biomolecules, such as nucleic acid molecules, proteins, lipids, carbohydrates, or combinations thereof. A tag can include one or more nucleic acid molecules, which can optionally encode information about the tag or the molecule to which the tag is conjugated (e.g., a binder, such as an antibody). For example, a tag can include a nucleic acid barcode molecule. A tag can include an organic compound or an inorganic compound.

[0087] As used herein, the term "barcode" generally refers to an identifying feature that can be used to distinguish between similar items. A barcode can comprise a nucleic acid molecule having from about 2 to about 30 bases. A barcode can comprise about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150 or more bases, which can provide a unique identifier tag or origin information for a molecule (e.g., a protein, polypeptide, peptide), a binder, a set of binders from a binding cycle, a sample molecule, a sample set, a molecule within a compartment (e.g., a droplet, bead, partition or separate location), a macromolecule within a set of compartments, a fraction of a macromolecule, a set of macromolecule fractions, a spatial region or a set of spatial regions, a macromolecule library or a binder library. A barcode can be an artificial sequence or a naturally occurring sequence, including peptides, proteins, protein complexes, carbohydrates and synthetic polymeric materials. In certain embodiments, each barcode within a barcode population is different. In other embodiments, a portion of the barcodes within the barcode population are different, e.g., at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% of the barcodes within the barcode population are different. A barcode population can be generated randomly or non-randomly. A barcode population can include error-correcting barcodes. Barcodes can be used to computationally deconvolve sequence reads derived from individual molecules, samples, libraries, etc. A barcode can contain, for example, multiplexing information generated by different samples, compartments, individual molecules, etc. Barcodes can also be used to deconvolve a collection of molecules that have been distributed into small compartments for enhanced mapping. For example, instead of mapping peptides back to a proteome, peptides can be mapped back to the protein molecule or protein complex from which they originated. The sample or partition, etc. from which it originated. A barcode can contain any useful sequence, including repetitive sequences (e.g., poly-A, poly-T, poly-C, poly-G regions), or a barcode can contain non-repetitive sequences.

[0088] As used herein, "sample barcode", also referred to as "sample label", generally refers to a barcode molecule that contains identification information of a sample, and the barcoded molecule is derived from the sample.

[0089] As used herein, "spatial barcode" generally refers to a barcode molecule that contains identification information of a 2D or 3D sample region (e.g., tissue section), and the molecule originates from or is derived from the region. Spatial barcodes can be used in molecular pathology on tissue sections. Spatial barcodes can allow multiplexed sequencing of multiple samples or libraries from tissue sections.

[0090] As used herein, "temporal barcode" generally refers to a barcode molecule that contains time-based information associated with the barcoded molecule. The types of time-based data encoded in a temporal barcode can include information such as the lifespan of the barcoded molecule, sample collection time, time or duration since the start of an experiment or induction by a stimulus, information about cell or tissue age, sequence of interactions between molecules, time or cycle or round (e.g., iterative process) providing the barcoded molecule, etc. It is possible to combine different types of barcodes (e.g., spatial, temporal, cell-specific) in a multiplexed barcode.

[0091] As used herein, the term "nucleic acid sequence" or "oligonucleotide sequence" generally refers to a continuous string of nucleotide bases, and can refer to the specific placement of nucleotide bases relative to each other when present in an oligonucleotide. Similarly, the term "polypeptide sequence" or "amino acid sequence" refers to a continuous string of amino acids, and can refer to the specific placement of amino acids relative to each other when present in a polypeptide.

[0092] The "nucleotide sequence" according to the present invention can include any polymer or oligomer of nucleotides, such as pyrimidine and purine bases, such as cytosine, thymine and uracil, and adenine and guanine, and combinations thereof. Nucleotide sequences can include any deoxyribonucleotide, ribonucleotide, hexitol nucleotide, cyclohexane nucleotide, peptide nucleic acid component and any chemical variant thereof, such as methylated, 7-deazapurine analogs, 8-halopurine analogs, hydroxymethylated or glycosylated forms of these bases, etc. These polymers or oligomers can be heterogeneous or homogeneous in composition, and can be isolated from naturally occurring sources or can be produced artificially or synthetically. In addition, nucleotide sequences can be DNA, RNA, HNA, CeNA or mixtures thereof, and can exist permanently or transiently in single-stranded or double-stranded form, including homoduplexes, heteroduplexes and hybrid states.

[0093] The terms "complementary" or "complementarity" refer to polynucleotides (i.e., nucleotide sequences) that are related by base pairing rules. For example, the sequence "5′-AGT-3′" is complementary to the sequence "5′-ACT-3′". Complementarity can be "partial", where only some of the bases of the nucleic acids match according to the base pairing rules, or there can be "complete" or "total" complementarity between the nucleic acids. The degree of complementarity between nucleic acid strands can have a significant effect on the hybridization efficiency and strength between the nucleic acid strands under defined conditions.

[0094] As used herein, the term "hybridization" is used in reference to the pairing of complementary nucleic acids. Hybridization and hybridization strength (e.g., the strength of the association between nucleic acids) are affected by factors such as the degree of complementarity between the nucleic acids, the stringency of the conditions involved, and the melting temperature of the hybrid formed. Hybridization methods involve annealing one nucleic acid to another complementary nucleic acid, e.g., based on Watson-Crick base pairing.

[0095] As used herein, the term "proteomics" generally refers to the quantitative and / or qualitative analysis of the proteome within a sample (such as a biological sample, e.g., from cells, tissues, or body fluids). Proteomics can include the analysis of the spatial distribution of proteins within a sample (e.g., cells and / or tissues). Proteomics can include the study of the dynamic state of the proteome, e.g., how one or more proteins change over time.

[0096] The terminal amino acid having a free amino group at one end of the peptide chain can be referred to herein as the "N-terminal amino acid" (NTAA). The terminal amino acid having a free carboxyl group at the other end of the chain can be referred to herein as the "C-terminal amino acid" (CTAA). The amino acids that make up the peptide can be numbered sequentially, where the length of the peptide is "n" amino acids. As used herein, in some cases, the NTAA can be considered the nth amino acid (also referred to herein as "n NTAA"). In such cases, the next amino acid is the n-1th amino acid, then the n-2th amino acid, and so on, decreasing the length of the peptide from the N-terminus to the C-terminus. Alternatively, the CTAA can be considered the nth amino acid (also referred to herein as "n CTAA"). In such cases, the next amino acid is the n-1th amino acid, then the n-2th amino acid, and so on, decreasing the length of the peptide from the C-terminus to the N-terminus. The NTAA, CTAA, or both can be modified or labeled with chemical moieties.

[0097] As used herein, the terms "determine", "measure", "evaluate", and "assess" are used interchangeably and include both quantitative and qualitative determinations.

[0098] As used herein, the term "unique molecular identifier" or "UMI" generally refers to a molecular barcode that contains indexing information. A UMI can comprise a nucleic acid molecule having a length of from about 3 to about 150 bases (3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145 or 150 bases). A UMI can provide a unique identifier tag for each molecule (e.g., peptide, binder, nucleic acid molecule) that contains the UMI or is coupled to the UMI. A UMI can include a random sequence (e.g., a random N-mer).

[0099] As used herein, a "derivative" of a nucleic acid molecule generally refers to a nucleic acid molecule that is derived from a source nucleic acid molecule. A derivative can have the same or substantially the same nucleotide sequence as the source nucleic acid molecule, or a derivative can comprise a complement or partial complement of the source nucleic acid molecule. A derivative can be the same type of nucleic acid as the source nucleic acid molecule (e.g., DNA or RNA), or a derivative can be a different type of nucleic acid (e.g., cDNA generated from an RNA molecule). A nucleic acid molecule derivative can exhibit sequence identity to the source nucleic acid molecule. Additional processing can also be performed on the derived nucleic acid molecule from the source nucleic acid molecule, e.g., chemical or enzymatic modification, splicing, ligation, polymerization, fragmentation, tagging (e.g., using a transposase), digestion, etc.

[0100] A derivative polypeptide or peptide can be derived from a source polypeptide (or peptide). A derivative can comprise the same amino acid sequence as the source polypeptide, or the sequence can be different. A derivative polypeptide can be produced from the source polypeptide or additional processing can be performed on the derivative polypeptide from the source polypeptide, e.g., chemical or enzymatic modification. A derivative polypeptide can include one or more tags, nucleic acid molecules, barcode molecules, labels (e.g., detectable labels), fluorophores, probes, linkers, post-translational modifications, chemical protecting groups or other chemical moieties.

[0101] As used herein, the term "compartment" generally refers to a physical region or volume that separates or isolates a subset of molecules from a molecular sample. For example, a compartment can separate individual cells from other cells, or separate a subset of the proteome of a sample from the remainder of the proteome of the sample. A compartment can be an aqueous compartment (e.g., a microfluidic droplet), a solid compartment (e.g., a plate, tube, vial, a picodroplet plate well or a microtiter well on a gel bead), or a separation region on a surface. A compartment can contain one or more beads to which macromolecules can be immobilized.

[0102] As used herein, the terms "solid support", "solid surface", or "solid substrate" or "substrate" refer to any solid material to which a molecule can associate directly or indirectly, including porous and non-porous materials. The molecule can associate with the substrate through covalent or non-covalent interactions or a combination thereof. The substrate can be two-dimensional (e.g., a planar surface) or three-dimensional (e.g., a gel matrix or beads). In non-limiting examples, the solid support can include beads, microbeads, arrays, glass surfaces, silicon surfaces, plastic surfaces, filters, membranes, nylon or other polymers, silicon wafer chips, flow-through chips, flow cells, biochips including signal transduction electronic devices, channels, microtiter wells, ELISA plates, rotating interferometric disks, nitrocellulose membranes, nitrocellulose-based polymer surfaces, polymer matrices, nanoparticles or microspheres. Materials used for the solid support include, but are not limited to, acrylamide, agarose, cellulose, nitrocellulose, glass, gold, quartz, polystyrene, poly(vinyl acetate), polypropylene, polymethacrylate, polyethylene, poly(ethylene oxide), polysilicate, polycarbonate, Teflon, fluorocarbon, nylon, silicone rubber, polyanhydride, poly(glycolic acid), poly(lactic acid), polyorthoester, functionalized silane, poly(propyl fumarate), collagen, glycosaminoglycan, polyamino acid, dextran, or any combination thereof. The solid support further includes thin films, membranes, bottles, disks, fibers, woven fibers, shaped polymers such as tubes, granules, beads, microspheres, microparticles, or any combination thereof. For example, when the solid surface is a bead, the bead can include, but is not limited to, ceramic beads, polystyrene beads, polymer beads, methylstyrene beads, agarose beads, acrylamide beads, solid beads, porous beads, paramagnetic beads, glass beads, or controlled pore beads. The bead can be spherical or irregular in shape. The size range of the bead can be from the nanoscale (e.g., 100 nm) to the millimeter scale (e.g., 1 mm). In certain embodiments, the size range of the bead is from about 0.2 microns to about 200 microns or from about 0.5 microns to about 5 microns.In some embodiments, the diameter of the beads can be about 1, 1.5, 2, 2.5, 2.8, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100 μm. In certain embodiments, the "bead" solid support can refer to a single bead or multiple beads.

[0103] As used herein, "sequencing" generally refers to determining: (A) the nucleotide (base sequence) order in a nucleic acid sample (e.g., DNA or RNA); or determining (B) the amino acid order in all or part of a polymer such as a protein, peptide, or other polymeric molecule. Many techniques are available, such as Sanger sequencing or high-throughput sequencing techniques (HTS). Sanger sequencing can involve sequencing via detection by (capillary) electrophoresis, in which up to 384 capillaries can be sequenced in one run. High-throughput sequencing involves parallel sequencing of thousands or millions or more sequences at a time. HTS can be defined as next-generation sequencing (NGS), i.e., next-next-generation sequencing techniques based on solid-phase pyrosequencing technology or single molecule real-time sequencing (SMRT). HTS technologies are available, such as those provided by Roche, Illumina, and Applied Biosystems (Life Technologies). Additional high-throughput sequencing techniques are described by and / or available from: Helicos, Pacific Biosciences, Complete Genomics, Ion Torrent Systems, Oxford Nanopore Technologies, Nabsys, ZS Genetics, GnuBio.

[0104] As used herein, "next-generation sequencing" refers to high-throughput sequencing methods that allow parallel sequencing of millions to billions of molecules. Examples of next-generation sequencing methods include sequencing by synthesis, sequencing by ligation, hybridization sequencing, polony sequencing, ion semiconductor sequencing, nanopore sequencing, and pyrosequencing. By attaching a primer to a solid substrate and attaching complementary sequences to nucleic acid molecules, the nucleic acid molecules can hybridize to the solid substrate via the primer, and then multiple copies can be generated in discrete regions on the solid substrate by amplification using a polymerase (these groupings are sometimes referred to as polymerase colonies or polonies). Thus, during the sequencing process, the nucleotides at a particular position can be sequenced multiple times (e.g., hundreds or thousands of times) - this depth of coverage is referred to as "deep sequencing". Examples of high-throughput nucleic acid sequencing technologies include platforms provided by Illumina, BGI, Qiagen, ThermoFisher, and Roche, including forms such as parallel bead arrays, sequencing by synthesis, sequencing by ligation, capillary electrophoresis, electronic microchips, "biochips", microarrays, parallel microchips, and single molecule arrays, as reviewed by Service (Science 311:1544-1546, 2006).

[0105] As used herein, "analyzing" a macromolecule generally refers to quantifying, characterizing, differentiating, or combinations thereof, of all or part of the components of a molecule (e.g., a macromolecule, a biomolecule such as a protein, an amino acid, a nucleic acid molecule, etc.). For example, analyzing a peptide, polypeptide, or protein can include determining all or part of the amino acid sequence (continuous or discontinuous) of the peptide. Analyzing a macromolecule can include partial identification of the components of the macromolecule. For example, partial identification of the amino acids in a protein sequence can identify the amino acids in the protein as belonging to a possible subset of amino acids. Analysis can be performed sequentially, e.g., starting with the analysis of nNTAA, and then proceeding to the next amino acid of the peptide (i.e., n-1, n-2, n-3, etc.). In such cases, sequencing can be performed by cleaving n NTAA, thereby converting the n-1th amino acid of the peptide to the N-terminal amino acid (referred to herein as "n-1NTAA"). Similarly, analysis of a peptide can start from the C-terminus towards the N-terminus, and each round of cleavage from the C-terminus generates a new CTAA. Cleavage of n CTAA converts the n-1th amino acid of the peptide to the C-terminal amino acid, referred to herein as "n-1CTAA". Analyzing a peptide can also include determining the presence and frequency of post-translational modifications on the peptide, which may or may not include information on the sequential order of post-translational modifications on the peptide. Analyzing a peptide can also include determining the presence and frequency of epitopes in the peptide, which may or may not include information on the sequential order or location of the epitopes within the peptide. Analyzing a peptide can include combining different types of analysis, e.g., obtaining epitope information, amino acid sequence information, post-translational modification information, or any combination thereof.

[0106] As used herein, the term "array" generally refers to a collection of molecules attached to one or more solid supports such that the molecules at one address can be differentiated from the molecules at other addresses. An array can include different molecules located at different addresses on a solid support. Alternatively, an array can include individual solid supports, each acting as an address bearing a different molecule, where the different molecules can be identified based on the position of the solid support on the surface to which the solid support is attached or based on the position of the solid support in a liquid (such as a fluid flow). The molecules of the array can be, for example, nucleic acids (such as SNAP), polypeptides, proteins, peptides, oligopeptides, enzymes, ligands, or receptors (such as antibodies, functional fragments of antibodies, or aptamers). The addresses of the array can optionally be optically observable, and in some configurations, neighboring addresses can be optically distinguishable when detected using the methods or devices described herein.

[0107] As used herein, the term "functionalized" refers to any material or substance that has been modified to include a functional group. The functionalized material or substance can be either naturally or synthetically functionalized. For example, a polypeptide can be naturally functionalized with a phosphate group, an oligosaccharide (such as a glycosyl, glycosylphosphatidylinositol, or phosphoglycosyl), a nitrosyl group, a methyl group, an acetyl group, a lipid (such as glycosylphosphatidylinositol, myristoyl, or isoprenyl), ubiquitin, or other naturally occurring post-translational modification. The functionalized material or substance can be functionalized for any given purpose, including altering chemical properties (such as changing hydrophobicity or changing surface charge density) or altering reactivity (such as being able to react with a moiety or reagent to form a covalent bond with that moiety or reagent).

[0108] As used herein, the terms “click reaction,” “click chemistry,” or “bioorthogonal reaction” refer to single-step, thermodynamically favorable conjugation reactions that utilize biocompatible reagents. Click reactions can be carried out without the use of toxic or biocompatibility-incompatible reagents (e.g., acids, bases, heavy metals) or without generating toxic or biocompatibility-incompatible by-products. Click reactions can use aqueous solvents or buffers (e.g., phosphate buffered solution, Tris buffer, saline buffer, MOPS, etc.). A click reaction can be thermodynamically favorable if it has a negative reaction Gibbs free energy, e.g., a reaction Gibbs free energy of less than about -5 kilojoules per mole (kJ / mol), -10 kJ / mol, -25 kJ / mol, -50 kJ / mol, -100 kJ / mol, -200 kJ / mol, -300 kJ / mol, -400 kJ / mol, or less than -500 kJ / mol. Exemplary bioorthogonal and click reactions are described in detail in WO 2019 / 195633A1, which is incorporated herein by reference in its entirety. Exemplary click reactions can include metal-catalyzed azide-alkyne cycloaddition, strain-promoted azide-alkyne cycloaddition, strain-promoted azide-nitrone cycloaddition, strain olefin reaction, thiol-ene reaction, Diels-Alder reaction, inverse electron demand Diels-Alder reaction, [3+2] cycloaddition, [4+1] cycloaddition, nucleophilic substitution, dihydroxylation reaction, thiol-yne reaction, photoclick reaction, nitrone dipole cycloaddition, norbornene cycloaddition, oxanorbornadiene cycloaddition, tetrazine ligation, and tetrazole photoclick reaction.Exemplary functional groups or reactive handles for performing click reactions can include alkenes (e.g., linear alkenes or cyclic alkenes such as trans-cyclooctene (TCO)), alkynes (e.g., linear alkynes or cycloalkynes (e.g., cyclooctyne or its derivatives such as dimethoxynitrilocyclooctyne (DIMAC), symmetric pyrrolo[2,1-c][1,2,5]triazin-4(8H)-one (SYPCO), pyrrolo[2,1-c][1,2,5]triazin-4(8H)-one (PYRROC), difluorocyclooctyne (DIFO), α,α-bis(trifluoromethyl)pyrrolo[2,1-c][1,2,5]triazin-4(8H)-one (TRIPCO), bicyclo[6.1.0]nonyne (BCN), dibenzocyclooctyne (DIBO), difluorocyclooctyne (DIFO), difluorobenzo[1,2-d:4,5-d']bis[1,3,2]dioxaborole (DIFBO), dibenzocyclooctyne (DBCO), difluorodiazadibenzocyclooctyne (F2-DIBAC), biaryl azacyclooctynone (BARAC), difluoromethoxydibenzocyclooctynol (FMDIBO), difluoromethoxydibenzocyclooctynone (keto-FMDIBO), and 3,3,6,6-tetramethylthiepin (TMTH)), TMTH-sulfoximine (TMTHSI), azides, epoxides, amines, thiols, nitrones, isonitriles, isocyanides, aziridines, activated esters, and tetrazines, triazoles, and combinations, variants, or derivatives thereof. The click chemistry moieties can be subjected to conditions sufficient to react a first click chemistry moiety with a second click chemistry moiety, e.g., providing a metal catalyst, a suitable solvent, pH, temperature, ionic concentration, or light / energy for any useful duration.

[0109] As used herein, the terms "group" and "moiety" are intended to be synonymous when used to refer to the structure of a molecule. The term refers to a component or part of a molecule. Unless otherwise indicated, the term need not indicate the relative size of the component or part compared to the molecule. Unless otherwise indicated, the term need not indicate the relative size of the component or part compared to any other component or part of the molecule. A group or moiety can contain one or more atoms.

[0110] As used herein, a "primer" generally refers to a nucleic acid molecule that can initiate the synthesis of a nucleic acid molecule (e.g., DNA or RNA). A primer can be single-stranded. A primer can contain one or more recognition sites for the binding of a protein (e.g., polymerase, restriction enzyme, cleavage enzyme, etc.) to the primer or to a template strand to which the primer hybridizes. A primer can include DNA, RNA, or other nucleic acid analogs or non-canonical bases (e.g., spacer moieties, uracil, abasic sites). A primer can optionally contain any number of functional sequences such as sequencing primer sequences (e.g., P5 or P7 sequences), sequencing primer binding sequences, read sequences (e.g., R1 or R2 sequences), restriction sites, transposon sites (e.g., chimeric end sequences), etc.

[0111] "Amplifying" or "amplify" generally refers to a polynucleotide amplification reaction, i.e., a population of polynucleotides replicated from one or more starting sequences. Amplification can refer to various amplification reactions, including but not limited to polymerase chain reaction (PCR), linear polymerase reaction, nucleic acid sequence-based amplification, rolling circle amplification, and similar reactions. The amplification reaction can generate amplicons.

[0112] As referred to herein, "adapter" generally refers to a short nucleic acid molecule (e.g., about 10 to about 100 base pairs in length). An adapter can include a short double-stranded DNA molecule. An adapter can be attached, for example, via polymerization or ligation to the end of a DNA fragment or amplicon. An adapter can include synthetic oligonucleotides, e.g., oligonucleotides having nucleotide sequences that are at least partially complementary to each other. An adapter can have blunt ends, can have staggered ends (also referred to herein as 3' or 5' "overhang sequences" or "sticky ends"), or both blunt ends and staggered ends. An adapter can be attached to a fragment (e.g., via ligation) to provide an adapter-ligated fragment; the adapter-ligated fragment can serve as a starting point for subsequent manipulation, e.g., for amplification or sequencing. An adapter can be functionalized, e.g., conjugated to a tag, probe, detectable label, affinity capture reagent (e.g., biotin or streptavidin).

[0113] As used herein, the terms "translocating" and "translocation" generally refer to the movement of a molecule through a medium (e.g., gas, liquid, or solid). Translocation of a molecule can occur spontaneously (e.g., by diffusion, Brownian motion, etc.). Alternatively or in addition, translocation of a molecule can occur by the application of a force or pressure, e.g., using frictional force, tension, normal force, air resistance, spring force, gravity, electric force, magnetic force, acoustic force (e.g., acoustophoresis), etc. In some examples, translocation of a molecule can be achieved by pressure-driven flow or electrophoretic force. Translocation can occur through a liquid or solid substrate (e.g., through a pore or gap).

[0114] As used herein, the abbreviations for natural 1-enantiomer amino acids are conventional and can be as follows: alanine (A, Ala); arginine (R, Arg); asparagine (N, Asn); aspartic acid (D, Asp); cysteine (C, Cys); glutamic acid (E, Glu); glutamine (Q, Gln); glycine (G, Gly); histidine (H, His); isoleucine (I, Ile); leucine (L, Leu); lysine (K, Lys); methionine (M, Met); phenylalanine (F, Phe); proline (P, Pro); serine (S, Ser); threonine (T, Thr); tryptophan (W, Trp); tyrosine (Y, Tyr); valine (V, Val). Unless otherwise specified, X can represent any amino acid. In some aspects, X can be asparagine (N), glutamine (Q), histidine (H), lysine (K), or arginine (R). These amino acids are also referred to in the form of "[amino acid][residue / residues]" (e.g., lysine residue, lysine residues, leucine residue, leucine residues, etc.).

[0115] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Suitable methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure.

[0116] Protein sequencing by coupling with polymerizable molecules

[0117] Methods, systems, compositions, and kits for characterizing proteins are provided herein. The methods, systems, compositions, and kits of the present disclosure are used to analyze individual amino acids of a protein, thereby providing information about the composition or sequence of amino acids in the protein (also referred to herein as "protein sequencing"). In some aspects, the methods of the present disclosure include providing an analyte (e.g., an amino acid) coupled to a polymerizable molecule and identifying the polymerizable molecule, the analyte (e.g., an amino acid), or both. In some aspects, the methods, systems, compositions, and kits provided herein require the use of nanoscale objects (e.g., nanopores or nanogaps) to identify amino acids that can be coupled to polymerizable molecules. Advantageously, the polymerizable molecule can alter or modulate the properties of the amino acid such that the amino acid is more accurately identifiable, e.g., translocating near or through a nanoscale object. Thus, the methods, systems, and compositions disclosed herein provide a simpler and more accurate method for protein sequencing.

[0118] In one aspect of the present disclosure, provided herein is a method for processing a modified amino acid, comprising: providing a modified amino acid, wherein the modified amino acid comprises a polymerizable molecule, and translocating the modified amino acid or its derivative through a nanopore or a nanogap. The method may further comprise measuring a signal from the nanopore or the nanogap and using the signal to determine the identity of the modified amino acid or its derivative. In some cases, the polymerizable molecule facilitates the translocation of the modified amino acid through the nanopore or the nanogap or alters the translocation rate of the modified amino acid through the nanopore or the nanogap, which may render the modified amino acid more readily detectable compared to an unmodified amino acid. In some cases, the translocation of the modified amino acid through the nanopore or the nanogap may improve the measured signal (e.g., current blockade) or increase the signal-to-noise ratio of the measured signal compared to an unmodified amino acid or an amino acid not conjugated to a polymerizable molecule.

[0119] Modified amino acid: The modified amino acid or its derivative may be derived from or be a part of a protein or a peptide; for example, the modified amino acid may comprise or be derived from an amino acid located at the peptide terminus (N-terminus or C-terminus), or the modified amino acid may comprise or be derived from an amino acid located within the peptide. The modified amino acid may comprise any number of modified proteinogenic amino acids or their derivatives. Examples of modifications include but are not limited to chemical modifications (e.g., protecting groups), biological modifications (e.g., post-translational modifications, modifications introduced by enzymatic treatment or digestion), physical modifications (e.g., mutations introduced by radiation, heating, etc.). In some cases, the modified amino acid or its derivative comprises or is conjugated to a binding agent, such as an antibody, an antibody fragment, a nanobody, an aptamer, a peptide, a small molecule, an inorganic compound, a polymer, or any variant or combination thereof. In some cases, the modified amino acid comprises a non-naturally occurring chemical modification. For example, the modified amino acid may comprise a protecting group, such as, in non-limiting examples, methyl, formyl, ethyl, acetyl, tert-butyl, anisyl, benzyl, trifluoroacetyl, N-hydroxysuccinimide, tert-butoxycarbonyl, benzoyl, 4-methylbenzyl, thiobenzoyl, thiotolyl, benzyloxymethyl, 4-nitrophenyl, benzyloxycarbonyl, 2-nitrobenzoyl, 2-nitrobenzenesulfinyl, 4-toluenesulfonyl, pentafluorophenyl, diphenylmethyl, 2-chlorobenzyloxycarbonyl, 2,4,5-trichlorophenyl, 2-bromobenzyloxycarbonyl, 9-fluorenylmethoxycarbonyl, triphenylmethyl, or 2,2,5,7,8-pentamethyl-chroman-6-sulfonyl. In some cases, the modified amino acid comprises a proteinogenic amino acid or its derivative conjugated to a polymerizable molecule. In some cases, the modified amino acid comprises a proteinogenic amino acid or its derivative, a linker, and a polymerizable molecule.

[0120] The modified amino acid can comprise any useful modification. The modification can be naturally occurring (e.g., post-translational modification) or non-naturally occurring, such as by labeling or tagging, e.g., with an amino acid or amine reagent or a linker comprising an amino acid or amine reagent. Examples of amino acid or amine reagents include isothiocyanates (e.g., PITC, NITC), 1-fluoro-2,4-dinitrobenzene (DNFB), dansyl chloride, 4-sulfonyl-2-nitrofluorobenzene (SNFB), acetylating agents, acylating agents, alkylating agents, guanidylating agents, thioacetylating agents, thioacylating agents, thiobenzoylating agents or derivatives or combinations thereof. Alternatively, or in addition, one or more modified amino acids can comprise adducts (e.g., polymers, such as PEG, polymerizable molecules, such as nucleic acid molecules, nanoparticles or nanotubes, peptides or proteins), lipids, carbohydrates, metabolites, fluorophores, haptens, quenchers, tags (e.g., fluorescent tags, magnetic tags, radioactive tags), barcodes or other moieties. In some cases, the modified amino acid can comprise a modification that facilitates recruitment of an enzyme (or ribozyme or deoxyribonuclease) to recognize or cleave the terminal amino acid (e.g., NTAA or CTAA) of a peptide. For example, the terminal amino acid of a peptide can be modified with a sugar to recruit a lectin or lectin-binding protease. In another example, one or more modified amino acids can comprise or be conjugated to a nucleic acid molecule having a first sequence that is complementary to a second sequence comprised by an oligonucleotide-binding protease. Hybridization of the first sequence to the second sequence can facilitate local recruitment of the protease to the amino acid to be cleaved. In another example, a peptide can be modified with phenylisothiocyanate (PITC), which can allow Edmanase recruitment and cleavage of the modified amino acid. In some examples, the modification of an amino acid can include an epitope tag, which can facilitate binding of a binder to the modified amino acid. Examples of such epitope tags include fluorophores, nucleic acid molecules, peptides, haptens, polymers, chemical moieties or other adduct molecules.

[0121] The methods described herein may also include generating modified amino acids. In some cases, the modified amino acids include proteinogenic amino acids or derivatives thereof, linkers, and polymerizable molecules. In one example, a peptide containing a terminal amino acid can be contacted with a linker comprising (i) a first reactive moiety capable of reacting with the amino acid and (ii) a second reactive moiety. Before, during, or after the reaction of the first reactive moiety with the terminal amino acid, a polymerizable molecule comprising a third reactive moiety capable of reacting with the second reactive moiety can be provided. The second and third reactive moieties can comprise click chemistry moieties capable of reacting with each other (e.g., azide and DBCO, azide and BCN, alkyne and DBCO, TCO and tetrazine, etc.). Thus, the reaction of the terminal amino acid with the linker and the reaction of the linker with the polymerizable molecule can produce a modified amino acid comprising the terminal amino acid, the linker, and the polymerizable molecule. In some cases, the terminal amino acid can be cleaved from the peptide, and the modified amino acid can comprise a cleavage product comprising the cleaved and optionally derivatized amino acid, the linker, and the polymerizable molecule.

[0122] Compared to unmodified amino acids or amino acids not conjugated with polymerizable molecules, the polymerizable molecule of the modified amino acid can alter the properties of the amino acid conjugated thereto; such altered properties (e.g., size, charge, aspect ratio, etc.) can affect the interaction or residence time of the modified amino acid with the nanopore or nanogap. In some embodiments, the polymerizable molecule alters the translocation speed of the modified amino acid when the modified amino acid passes through the nanopore or nanogap compared to the translocation speed of the unmodified amino acid. The translocation speed of the modified amino acid can be higher or lower than the translocation speed of the unmodified amino acid. In some cases, the translocation speed of the modified amino acid is lower than the translocation speed of the unmodified amino acid. For example, the translocation speed of the modified amino acid can be reduced by at least about 0.1%, at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 20%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 200%, at least about 500%, at least about 1000%, at least about 5000%, or more. The translocation speed of the modified amino acid can be reduced by a certain percentage range, such as about 5% - 20%, about 500% - 3000%, etc. In some cases, the translocation speed of the modified amino acid is non-uniform, e.g., can fall within a certain percentage range of translocation speed variation, and can depend on the nature of the polymerizable molecule, the amino acid comprised in the modified amino acid, the linker, or any other component of the molecule (e.g., size, charge, polarity, etc.).

[0123] Compared with unmodified amino acids (e.g., amino acids not conjugated to polymerizable molecules), changes in the translocation speed of modified amino acids can make the modified amino acids more readily detectable. For example, a polymerizable molecule conjugated to an amino acid can reduce or alter the translocation speed as it passes through a nanopore or nanogap, enabling a more accurate current reading. Alternatively or in addition, the polymerizable molecule can alter the charge, size, aspect ratio of the amino acid to which it is conjugated, which can change the current signature to enhance the detectability of the amino acid, the polymerizable molecule, or both. Alternatively or in addition, the medium in contact with the nanopore (e.g., liquid, buffer, solution) can contain one or more reagents that can change or modulate the translocation speed of the modified amino acid. For example, compared with unmodified amino acids, the medium can contain ions, salts, or other molecules that can selectively or preferentially alter the interaction between the modified amino acid and the nanopore.

[0124] In some cases, when a modified amino acid passes through a nanopore or nanogap, the measured signal-to-noise ratio (SNR) is higher than that of an unmodified amino acid (e.g., a protein-native amino acid, an amino acid without a conjugated polymerizable molecule). The increase in SNR can be at least about 0.1%, at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 20%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 200%, at least about 500%, at least about 1000%, at least about 5000%, or greater. The SNR of the modified amino acid can increase within a certain percentage range, such as about 5% - 20%, about 500% - 1000%, etc.

[0125] In some cases, the signal measured (e.g., current blockade) when a modified amino acid passes through a nanopore or nanogap is significantly different from that of an unmodified amino acid. In some cases, the signal measured when a modified amino acid passes through a nanopore or nanogap is significantly different from that of the polymerizable molecule alone. The difference in the measured signal can be measured by any useful metric, such as fold change, percent change, absolute current measurement, signal-to-noise ratio, etc.

[0126] The polymerizable molecule can comprise any useful polymerizable moiety. The polymerizable moiety can comprise a naturally occurring or synthetic polymer (organic or inorganic) or a biopolymer. The polymer can comprise one or more monomer types. In the case of using more than one monomer type, the polymer can form an alternating copolymer structure, a periodic copolymer structure, a random copolymer structure, a block copolymer structure, a chain or graft copolymer, or any other useful structure. The polymer can be linear or non-linear. The polymer can comprise polyethylene glycol (PEG), PEG-diacrylate, PEG-acrylate, PEG-thiol, PEG-azide, PEG-alkyne, polyacrylamide, agarose, collagen, fibrin, gelatin, chitosan, hyaluronic acid, alginate, polyvinyl alcohol, or other polymers.

[0127] In some examples, the polymerizable molecule comprises a biomolecule, such as a protein or a peptide, a nucleic acid molecule, such as DNA or RNA, a carbohydrate, or a lipid chain. In some cases, the polymerizable molecule comprises a nucleic acid molecule, which can comprise any useful number and type of nucleotides, such as including canonical and non-canonical bases, and the number of nucleotides can be adjusted according to the intended purpose. For example, the length of the nucleic acid molecule can be adjusted to change the properties (such as volume, aspect ratio, charge, etc.) of the amino acid tethered to the polymerizable molecule. The nucleic acid molecule can additionally or alternatively comprise any useful functional sequences, including but not limited to barcode sequences or other recognition sequences, UMI sequences, enzyme recognition sites (such as transposon sites, restriction sites), spacer sequences, sequencing primer sequences, read sequences, or primer sequences. The nucleic acid molecule can comprise canonical bases, non-canonical bases, natural bases, synthetic bases, abasic sites, or combinations thereof. In some cases, modified amino acids or their derivatives are generated using an iterative process, as described elsewhere herein; thus, the nucleic acid molecule can comprise information about the number of rounds or cycles of the iterative process. In some cases, the nucleic acid molecule comprises a nucleic acid barcode molecule and can comprise useful information about amino acid identity, temporal information, spatial information, etc.

[0128] Polymerizable molecules (e.g., nucleic acid molecules) can be coupled to amino acids covalently or non-covalently. The coupling can be carried out using any suitable chemistry and reaction conditions and can include the use of linkers. In one example, a nucleic acid molecule can include a first reactive group, e.g., a first click chemistry moiety as described elsewhere herein, and can be contacted with a linker that includes a second reactive group, e.g., a second click chemistry moiety capable of reacting with the first reactive group. The linker can also include additional reactive groups capable of tethering to an amino acid (e.g., a terminal amino acid) and optionally cleaving the amino acid from a peptide. For example, the additional reactive group can be a thiocyanate conjugate (e.g., isothiocyanate (ITC) (such as phenyl isothiocyanate (PITC) or naphthyl isothiocyanate (NITC))) or an aldehyde group (e.g., ortho-phthalaldehyde (OPA), 2,3-naphthalenedialdehyde (NDA)), guanidinating agent, dinitrofluorobenzene (DNFB), dansyl chloride, or other amino acid reactive groups. The linker can react with an amino acid of the peptide, e.g., NTAA or CTAA. Using such linkers that contain at least two reactive groups can allow (i) tethering of the amino acid to the linker and (ii) tethering of the linker to the nucleic acid molecule (see, e.g., Figure 4 ). In some cases, the linker can be pre-tethered to the nucleic acid molecule prior to contacting with the amino acid. In some cases, the conjugation of the polymerizable molecule to the amino acid (whether through a linker or without a linker) can alter the chemical structure of the amino acid. For example, if a linker containing an isothiocyanate moiety is used, the amino acid can be derivatized to a thiocarbamoyl group (e.g., under basic conditions), a thiazolone group (e.g., under acidic conditions), a thiohydantoin group, or other chemical moieties, thereby producing a modified amino acid that contains the polymerizable molecule conjugated thereto.

[0129] Linkers: One or more linkers can be used to couple a polymerizable molecule to an amino acid or its derivative, thereby generating a modified amino acid. In some cases, the linker contains a click chemistry moiety. The click chemistry moiety can include any suitable bioorthogonal moiety as described elsewhere herein, e.g., alkenes, alkynes (e.g., alkynes, cycloalkynes, e.g., DBCO and BCN), azides, epoxides, amines, thiols, nitrones, isonitriles, isocyanides, aziridines, activated esters, and tetrazines, as well as combinations, variants, or derivatives thereof. The linker can be subjected to conditions sufficient to cause the first click chemistry moiety to react with the second click chemistry moiety, e.g., providing a metal catalyst, a suitable solvent, pH, temperature, ionic concentration, or light / energy for any useful duration.

[0130] The linker can comprise an amino acid-reactive moiety. The amino acid-reactive moiety of the linker can be any useful moiety that enables the reactive moiety to conjugate with an amino acid and optionally cleave the amino acid. In some examples, a third reactive moiety can react with a terminal amino acid (e.g., NTAA or CTAA). In such examples, the third reactive moiety can comprise any primary amine or carboxyl-reactive group, including but not limited to isocyanates, acyl azides, NHS esters, sulfonyl chlorides, aldehydes, glyoxals, epoxides, ethylene oxides, carbonates, aryl halides, imido esters, carbodiimides, acid anhydrides, phenyl esters, isothiocyanates (e.g., phenyl isothiocyanate, sodium isothiocyanate, ammonium isothiocyanate (e.g., tetrabutylammonium isothiocyanate, tetrabutylammonium isothiocyanate), diphenylphosphoryl isothiocyanate), acetyl chloride, cyanogen bromide, carboxypeptidase, azides, alkynes, DBCO, maleimides, succinimides, thiol-thiol disulfide bonds, tetrazines, TCO, vinyls, methylcyclopropenes, acryloyls, allyls, etc. Additional examples of amino acid-reactive groups are provided in U.S. Patent Publication No. 2020 / 0217853, which is incorporated herein by reference in its entirety.

[0131] The linker can comprise any additional useful moiety. For example, the linker can comprise a releasable or cleavable moiety, which can facilitate removal of the amino acid-linker complex from the polymerizable molecule or a portion thereof or from the substrate to remove monomers. Such a releasable or cleavable moiety can comprise, for example, a disulfide bond, which can be released by contact with a reducing agent (e.g., DTT, TCEP). In some examples, the linker can be coupled to an intermediate linking molecule via a releasable or cleavable moiety, alternatively or additionally via click chemistry coupling. Thus, the coupling between the intermediate linking polymerizable molecule and the linker can be reversible. Additionally, the linker can comprise any number of spacer moieties, such as polymers (e.g., PEG, PVA, polyacrylamide), 6-aminohexanoic acid, nucleic acids, alkyl chains, etc. Such spacer moieties can increase the distance between any other moieties of the linker (e.g., amino acid-reactive groups and polymerizable molecule-reactive groups).

[0132] In some cases, the polymerizable molecule comprises a linker. The linker can be used, for example, to couple a reactive moiety to the polymerizable molecule, and the reactive moiety can react with the reactive moiety of another linker. In one such example, the polymerizable molecule (e.g., a nucleic acid molecule) can comprise a linker that contains a click chemistry moiety. The linker containing the click chemistry moiety can be coupled to the polymerizable molecule using any useful method (e.g., by incorporating linker-conjugated nucleotides or nucleosides), and can be located at any useful position (e.g., at the 5'-end, 3'-end, center of the polymerizable molecule). For example, click-functionalized nucleotides or nucleosides (e.g., ethynyl deoxyuridine, octadiynyl deoxyuridine) can be incorporated into the backbone of a DNA or RNA molecule. In this way, the click chemistry moiety of the polymerizable molecule can be coupled to another linker that contains a complementary click chemistry moiety and an amino acid reactive group (e.g., isothiocyanate, dansyl chloride, DNFB, etc.).

[0133] The linker can comprise any number of spacer moieties, such as alkyl chains, polymeric spacers (e.g., PEG), nucleic acid or oligomeric spacers, or other useful spacer moieties that can be used to adjust the size or molecular weight of the linker. For example, the linker can comprise at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10 or more spacer moieties (e.g., hydrocarbon units, PEG units, nucleotides or spacer sequences, etc.). The linker can comprise up to about 100, up to about 10, up to about 9, up to about 8, up to about 7, up to about 6, up to about 5, up to about 4, up to about 3, up to about 2 or up to 1 spacer moiety. The linker can comprise any useful number of functional groups, such as for attaching multiple molecules. The linker can comprise at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10 or more functional groups.

[0134] Nanopore / nanogap: Modified amino acids can be characterized using nanoscale techniques such as nanopores, nanogaps or nanochannels. In some cases, nanopores, nanogaps or nanochannels can be provided in a membrane in an ionic solution. Signals can be measured from the nanopore, the membrane or the surrounding solution. For example, conductance, current, current blockage or other parameters within the nanopore can be monitored as a function of time. When a molecule enters the nanopore, nanogap or nanochannel, the conductance, current or other parameters can change and provide information about the molecule (e.g., size, charge, aspect ratio, volume). Each amino acid or modified amino acid, or a subset of amino acids or modified amino acids, can produce a unique signal that can be distinguished from other amino acids or modified amino acids. Thus, unique signal signatures can be assigned to amino acids or modified amino acids to determine the identity of the amino acid, the polymerizable molecule, or both.

[0135] In some cases, the modified amino acid can be analyzed multiple times, e.g., by translocation through the same or different nanopores or nanogaps and measuring the current or conductance of the modified amino acid. For example, iterative reads of the modified amino acid can facilitate improved accuracy of readout or identification of the modified amino acid (or polymerizable molecule). In such cases, the modified amino acid can translocate through one or more nanopores at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 20 times, at least 50 times, at least 100 times, at least 200 times or even more. Alternatively or additionally, the modified amino acid can move back and forth within the nanopore, e.g., using a processive enzyme such as phi29 polymerase, e.g., as described in Cherf et al. 2012. Nat. Biotechnol. 30(4):344-348, which is incorporated herein by reference in its entirety.

[0136] Nanopores, nanogaps or nanochannels can be generated from organic materials (e.g., pore-forming proteins or transmembrane proteins). Such proteins can be naturally occurring, synthetic or engineered. Examples of naturally occurring organic nanopores include wild-type aerolysin, α-hemolysin, mycobacterial porins (e.g., MspA porin), Phi29 connector channel, Fragaceatoxin C, cytolysin A, ferric hydroxamate uptake component A, Curli-specific gene G, outer membrane porin G, viral DNA packaging motor, etc. Alternatively or additionally, nanopores, nanochannels or nanogaps can include engineered variants of naturally occurring nanopores. In some embodiments, the nanopore, nanogap or nanochannel consists of an inorganic material. For example, solid-state nanopores can be made from dielectric materials such as silicon compounds (e.g., silicon nitride, silicon dioxide), aluminum compounds (e.g., aluminum oxide), titanium compounds (e.g., titanium oxide), molybdenum compounds (e.g., molybdenum sulfate), hafnium, graphene, etc. Nanopores, nanochannels or nanogaps can adopt any useful shape factor or geometry, e.g., a gap or channel within a membrane, capillary, etc., and can be generated using any suitable process, e.g., ion beam milling, electron beam lithography. Nanopores, nanochannels or nanogaps can include elastic materials.

[0137] In some cases, a nanopore, nanogap, or nanochannel is conjugated with a protein. The protein can be a molecular motor that can facilitate the movement of a modified amino acid or a portion thereof (e.g., a polymerizable material) through the nanopore. In a non-limiting example, the modified amino acid can include an amino acid optionally conjugated to a nucleic acid molecule via a linker, and the nanopore can be conjugated with a helicase or a molecule containing helicase activity. The helicase can be used to translocate the nucleic acid molecule and the conjugated amino acid through the nanopore. In some cases, compared to an unmodified amino acid, the molecular motor can increase, decrease, or otherwise alter the translocation rate of the modified amino acid through the nanopore. In other examples, the molecular motor can include a topoisomerase, polymerase, nuclease (e.g., an endonuclease such as a restriction endonuclease or a Cas protein, or an exonuclease), unfoldase, mitotic spindle protein (e.g., nuclear mitotic apparatus protein, kinesin, dynein), or other motor proteins (e.g., myosin), variants thereof, or other polymer processing proteins. In certain cases, the protein includes a protease or proteasome that can effect "chop-n-drop" or cleavage of the modified amino acid or a portion thereof prior to translocation through the nanopore.

[0138] Alternatively or in addition, translocation of the molecules described herein (e.g., modified amino acids, polymerizable molecules, etc.) through a medium or through a nanopore or nanogap can be facilitated by applying a force. For example, molecules can be translocated by applying pressure (e.g., pressure-driven flow), an electric field (e.g., by electrophoresis, electroosmotic flow, isoelectric focusing), a magnetic field (e.g., using ferrofluids, magnetic particles), or light (e.g., optoelectronics).

[0139] Commercially available nanopore systems can be used in the methods described herein. For example, nanopore systems from Oxford Nanopore Technologies (ONT), such as the MinION, VolTRAX, GridION, PromethION, MinIT, Flongle, or Q-Line products, can be used to identify and characterize the modified amino acids described herein.

[0140] Intramolecular expansion: The intramolecular expansion method can be used to generate modified amino acids or their derivatives, for example, using one or more linkers and polymerizable molecules. In intramolecular expansion, individual amino acids, amino acid clusters, or small peptides (such as dipeptides, tripeptides, or tetrapeptides) of a protein (such as a peptide or protein analyte) can be sequentially removed and re-tethered together so that the distance between individual amino acids or amino acid clusters is increased. Advantageously, the intramolecular expansion process on one or more amino acids of a peptide can avoid or overcome several problems of peptide nanopore sequencing. For example, by increasing the spacing between amino acids, fewer amino acids can enter the nanopore in a given situation, thereby reducing the number of superimposed signals caused by the number of amino acids within the nanopore. In addition, increasing the spacing between amino acids can disrupt the intramolecular interactions of the convolution current blocking signal, thereby allowing a higher-precision signal to be output from the nanopore.

[0141] In one example, a method of in-molecule amplification can include providing a peptide comprising a plurality of amino acids, a linker (e.g., as described elsewhere herein), a polymerizable molecule (e.g., as described elsewhere herein), and a capture moiety. The linker can be configured to couple to (i) an amino acid of the peptide (e.g., NTAA or CTAA) and (ii) the polymerizable molecule. The method can also include contacting the linker with the amino acid and the polymerizable molecule. Alternatively, or in addition, the linker can be pre-tethered to the polymerizable molecule and then reacted with the amino acid. The linker can couple to the amino acid of the peptide to produce an amino acid-linker complex. The amino acid-linker complex can then be coupled to the capture moiety via the polymerizable molecule. For example, both the polymerizable molecule and the capture moiety can include nucleic acid molecules, which can be coupled by hybridization, ligation, or both. In some cases, the method can also include coupling the polymerizable molecule to the capture moiety, cleaving the amino acid from the peptide to produce an amino acid-linker-capture moiety (AALC) complex, and optionally repeating the process. In an example of repeating the process, an additional linker can be provided, which is configured to couple to (i) an additional amino acid of the peptide (e.g., n-1NTAA or n-1CTAA) and (ii) an additional polymerizable molecule. The method can also include contacting the additional linker with the additional amino acid to produce an additional amino acid-linker complex. The additional polymerizable molecule can be coupled to the linker before, during, or after the linker is coupled to the additional amino acid. The additional polymerizable molecule can be configured to couple to the AALC complex (e.g., via the polymerizable molecule of the AALC complex). Thus, in some examples, after generating the additional linker-additional amino acid complex, the additional linker-additional amino acid complex can be coupled to the AALC complex, thereby generating a stacked AALC complex, and the additional amino acid can be cleaved from the peptide before, during, or after the stacked AALC complex is generated. Thus, a "modified amino acid" can refer to an amino acid-linker complex, an amino acid-linker-polymerizable molecule, an AALC complex, a stacked AALC complex (comprising two or more amino acids), or a cleaved AALC complex, or a combination or portion thereof (e.g., only the amino acid portion included, only the amino acid-linker complex portion, etc.).

[0142] In some cases, intramolecular amplification of a peptide or protein can occur on multiple capture moieties. For example, the use of a substrate can facilitate intramolecular amplification of a substrate-binding peptide. For example, a substrate comprising multiple capture moieties can be provided, and in some cases, the capture moieties are located near the peptide or protein. The first amino acid of the peptide or protein (e.g., n NTAA or n CTAA) can be coupled to the first capture moiety (e.g., via a first linker and a first polymerizable molecule), the second amino acid (e.g., n-1NTAA or n-1CTAA) can be coupled to the second capture moiety (e.g., via a second linker and a second polymerizable molecule), and the third amino acid (e.g., n-2NTAA or n-2CTAA) can be coupled to the third capture moiety (e.g., via a third linker and a third polymerizable molecule). In another example, the first amino acid (e.g., n NTAA) can be coupled to the first capture moiety, the second amino acid (e.g., n-1NTAA) can be coupled to an AALC complex (e.g., from n NTAA), thereby generating a stacked AALC complex, and the third amino acid (e.g., n-2NTAA) can be coupled to the second capture moiety. It should be understood that any number of amino acids (or modified amino acids) can be coupled to any number of capture moieties (or resulting AALC or stacked AALC complexes).

[0143] Capture moiety: A capture moiety can be coupled to an amino acid, a linker, or a polymerizable molecule. The coupling of an amino acid, a linker, or a polymerizable molecule to a capture moiety can include covalent interactions or non-covalent interactions. The coupling can occur through the interaction of a binding pair, for example, biotin and avidin (or streptavidin), an antigen or epitope and an antibody or antibody fragment, cyclodextrin and a hydrophobic small molecule (e.g., alkane, benzene, polycyclic compound), cucurbituril and adamantylammonium or trimethylammonium ferrocene, diphenylene cycloalkane (e.g., calixarene, cavitand, pillararene, tetralactam), etc. In some embodiments, the coupling of an amino acid, a linker, or a polymerizable molecule to a capture moiety occurs through the coupling of nucleic acid molecules (e.g., hybridizing to each other or to a splint molecule).

[0144] In some cases, the capture moiety includes an additional polymerizable molecule (e.g., a nucleic acid molecule or a peptide). In one such example, both the polymerizable molecule of the modified amino acid and the capture moiety can include nucleic acid molecules. These nucleic acid molecules can be coupled to each other, for example, directly by complementary base pairing, or by a splint molecule and an optional linker.

[0145] The nucleic acid molecules of the capture moiety can include any naturally occurring, non-naturally occurring, or engineered nucleobases. For example, the nucleic acid molecules can include pseudo-complementary bases, bridged nucleic acids, xeno nucleic acids, locked nucleic acids, peptide nucleic acids (PNAs), γ-PNAs, morpholinos, etc., as described elsewhere herein. The capture moiety can include one or more functional sequences, including but not limited to primer sequences, sequencing sequences (e.g., P5 or P7 sequences), sequencing read sequences (e.g., R1 or R2 sequences), chimeric end sequences, transposase recognition sequences, cleavage sites (e.g., restriction sites), UMIs, blocking groups, spacer sequences, barcode sequences, or other functional sequences. In some cases, the capture moiety includes a cleavable or releasable moiety, e.g., a restriction enzyme recognition site, an abasic site, uracil that can be cleaved using or uracil DNA glycosylase, a disulfide bond that can be released upon addition of a reducing agent, etc. In some cases, the capture moiety contains a partial restriction site; for example, the capture moiety can contain a first partial restriction site, while the polymerizable molecule can contain a second partial restriction site; after the polymerizable molecule is coupled or linked to the capture moiety, the two partial restriction sites can generate a complete restriction site such that a single molecule (the capture moiety and the polymerizable molecule) cannot be cleaved alone by restriction digestion, but the product of the ligation or coupling can. In some cases, the capture moiety contains a barcode sequence that contains any useful information, e.g., the identity of the peptide to be analyzed, time information, spatial information, etc.

[0146] In some cases, the capture moiety is coupled to a substrate. In one example, the substrate contains one or more identical capture nucleic acid molecules; these identical capture nucleic acid molecules can serve as the capture moiety for generating one or more AALC complexes, e.g., for terminal amino acids, n-1 amino acids, etc. In some cases, commercially available substrates can be used, such as beads (e.g., DNA beads or barcoded beads), flow cells, or chips, e.g., HiSeq, iSeq, MiniSeq, NextSeq, NovaSeq, etc. as the substrates described herein. In some cases, the capture moiety can contain other useful sequences, such as primer sequences (e.g., P5 or P7 sequences) or read sequences (e.g., R1 or R2).

[0147] The capture moiety can be coupled to the substrate using any useful method. In some cases, the capture moiety includes a substrate tethering group or linker or additional functional groups. In some examples, the capture moiety includes a nucleic acid molecule that contains a substrate tethering group (e.g., biotin), a click chemistry moiety (such as an azide) that can couple to the substrate (e.g., a substrate containing streptavidin), or a complementary click chemistry moiety that can react with the substrate tethering group. The capture moiety can additionally include a binding sequence to which another nucleic acid molecule (e.g., as part of a modified amino acid or a polymerizable molecule conjugated to a modified amino acid) can bind. In some cases, the capture moiety includes a single-stranded oligonucleotide or a single-stranded region in which complementary oligonucleotides can hybridize. The complementary oligonucleotide can include a detectable label (e.g., a fluorophore) that allows detection of the capture moiety.

[0148] Alternatively, the capture moiety can be uncoupled from the substrate. For example, the capture moiety can be directly conjugated to a peptide to be analyzed or undergoing in-molecule amplification. In such an example, the capture moiety can additionally include a nucleic acid barcode molecule that encodes the identity of the peptide or the original sample or partition from which the peptide is derived.

[0149] Cleavage: In some cases, the method further includes cleaving an amino acid or a modified amino acid from the peptide. Cleaving an amino acid or a modified amino acid from a polymeric analyte can be achieved using any suitable mechanism (such as via application of a stimulus). The stimulus can be, for example, a chemical stimulus, a biological stimulus, a thermal stimulus (e.g., application of heat), a light stimulus, a physical or mechanical stimulus, or other types of stimuli or a combination of stimuli. In some cases, the stimulus includes a chemical stimulus, such as a change in pH, the application of an acid or a base, the addition of a solvent, an initiator, a radical generator, a reducing agent, etc. In some cases, the chemical stimulus includes the application of a Lewis acid (e.g., boron trifluoromethanesulfonate, boron trifluoride diethyl etherate, boron trichloride, boron tribromide, boron triiodide, or scandium trifluoromethanesulfonate). In some cases, the stimulus includes a biological stimulus, such as an enzyme (e.g., Edmanase, protease, endonuclease) or a ribozyme or deoxyribozyme that can cleave or catalyze the cleavage of a modified amino acid from the polymeric analyte.

[0150] In some examples, the method can include using a linker that includes an amino acid reactive group (e.g., PITC), coupling the amino acid reactive group of the linker to an amino acid, and using a stimulus (e.g., change in pH, temperature) to cleave the amino acid from the peptide. In an example, the linker includes a PITC moiety that can couple with an NTAA under mild basic conditions to generate a phenylthiocarbamoyl (PTC) derivative of the NTAA, and cleavage of the NTAA from the peptide can be achieved using an Edman degradation reaction (e.g., a polymerizable molecule, such as a nucleic acid molecule) to produce a thiazolinone (ATZ) derivative or a phenylthiohydantoin (PTH) derivative. As described elsewhere herein, the linker can include a moiety or molecule (e.g., a nucleic acid molecule or a polymerizable molecule) that can also be coupled to a capture moiety such that the amino acid or modified amino acid can be coupled to the capture moiety, thereby generating an AALC complex.

[0151] Given the harsh reaction conditions of standard Edman degradation, the polymerizable molecules (e.g., nucleic acid molecules, peptides, lipids) etc. described herein can include alterations or modifications to make them more resistant to the reaction conditions. For example, a nucleic acid molecule can predominantly include pyrimidines (e.g., thymine, cytosine, uracil) which are more resistant to acid degradation and heat compared to purines (e.g., adenine and guanine). For example, a nucleic acid molecule can include at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100% thymine or cytosine. Alternatively or additionally, typical nucleotides can be substituted or can include acid-resistant nucleotide analogs, such as hexitol nucleic acid.

[0152] Alternative degradation chemistries can also be employed. Mild degradation for removing N-terminal amino acids under basic conditions can include using triethylammonium acetate in acetonitrile or other solvents such as water, N,N-dimethylformamide (DMF) or solvent mixtures. Alternatively, degradation can be achieved using a thioacylation method, using milder acid reagents such as trichloroacetic acid (pKa of 0.66) or dichloroacetic acid (pKa of 1.35), or using alternative basic reaction conditions, such as using an acid-base pair, such as N,N-diisopropylethylamine (DIPEA), pyridine, acetic acid derivatives, etc.

[0153] The present invention also provides C-terminal degradation strategies. C-terminal degradation can include Edman-like degradation methods. C-terminal degradation can employ activating and derivatizing reagents (e.g., thiocyanates to generate peptide-thiocyanates or peptide-thiohydantoins) that react with the C-terminal carboxyl group of the peptide. Non-limiting examples of activating reagents include acetyl chloride and acetic anhydride. Alternatively or additionally, one-step C-terminal derivatization of a peptide to a peptidyl thiohydantoin can be carried out, for example, using the Schlack-Kumpf method, in which the peptide is reacted with thiocyanic acid (e.g., in acetone) to generate a peptidyl thiohydantoin. The peptide-thiohydantoin can be cleaved, for example, using basic conditions, to generate amino acid thiohydantoins and the remaining peptide.

[0154] Cleavage of amino acids can also be achieved using enzymatic or enzyme mimetic (e.g., ribozyme or deoxyribozyme) methods. Example enzymatic cleavage can include using Edmanase (e.g., modified cruzain), aminopeptidases (e.g., Pfu aminopeptidase I, PhTET aminopeptidase, Pyrococcus horikoshii aminopeptidase), metalloenzymatic aminopeptidases, acylpeptide hydrolases, tRNA synthetases, endopeptidases, carboxypeptidases, etc. The enzyme or ribozyme or deoxyribozyme can be modified or engineered to recognize modified amino acids, e.g., amino acids having a chemical moiety attached thereto (e.g., PITC, NITC, dansyl chloride, SNFB, DNP, SNP, biotin, streptavidin, nucleic acid molecule, lipid, carbohydrate, acetyl group, acyl group, guanylating agent, etc.).

[0155] One or more reactions can be accelerated by applying energy or radiation such as electromagnetic radiation. For example, degradation or cleavage of the terminal amino acids of a peptide can be facilitated by accelerating the reaction kinetics by applying microwave energy. For example, hydrolysis of a protein can be facilitated by applying microwave energy, e.g., as described in Margolis et al., 1991, Journal of Automatic Chemistry. Vol 13, No. 3, pp 93-95 (which is incorporated herein by reference).

[0156] In some cases, each cleavage event can cleave more than one amino acid from a peptide. Cleavage can include cleavage of 2 amino acids, 3 amino acids, 4 amino acids, 5 amino acids, 6 amino acids, 7 amino acids, 8 amino acids, 9 amino acids, 10 amino acids, or more. For example, a polymeric analyte can include a peptide containing multiple amino acids, and single amino acids, dipeptides, tripeptides, tetrapeptides, or larger peptides can be cleaved in the methods described herein. In some cases, up to about 10 amino acids, up to about 9 amino acids, up to about 8 amino acids, up to about 7 amino acids, up to about 6 amino acids, up to about 5 amino acids, up to about 4 amino acids, up to about 3 amino acids, or fewer amino acids can be cleaved in a given cleavage event. In some cases, an enzyme (e.g., Edmanase, protease) or ribozyme or deoxyribozyme capable of recognizing or cleaving multiple amino acids can be used to mediate cleavage of multiple amino acids.

[0157] Cleavage of amino acids or modified amino acids can be performed using a biological stimulus such as an enzyme or ribozyme or deoxyribozyme. The enzyme can be any useful cleavage enzyme, e.g., a protease such as Edmanase, cruzain, a clipping protein (e.g., ClpS, ClpX), proteinase K, an exopeptidase, an aminopeptidase, a diaminopeptidase, a serine protease, a cysteine protease, a threonine protease, an aspartic protease, an aspartic protease, a glutamic protease, a metalloprotease, an asparaginyl peptide cleaving enzyme, pepsin, trypsin, pancreatin, Lys-C, Glu-C, Asp-N, chymotrypsin, a carboxypeptidase (e.g., carboxypeptidase A, carboxypeptidase B, carboxypeptidase Y), a SUMO protease, elastase, papain, an endoprotease, a protease, Bromelain, collagenase, hyaluronase, thermolysin, ficin, keratinase, tryptase, fibroblast activation, enterokinase, chymotrypsinogen, gastrokine, clostripain, calpain, alpha-lytic protease, proline-specific endopeptidase, furin, thrombin, subtilisin, genenase, PCSK9, cathepsin, aminoprolinase, methionine aminopeptidase, cathepsin C, 1-cyclohexen-1-yl-boronic acid pinacol ester, pyroglutamate aminopeptidase, renin, kininogen, kallikrein, DPPIV / CD26, thimet oligopeptidase, prolidase, leucine aminopeptidase, dipeptidyl peptidase or other enzyme or protease, or a combination or variant thereof (e.g., engineered mutant or variant). In some cases, a cleavage enzyme or ribozyme or deoxyribozyme can be configured or engineered to cleave terminal amino acid or amino acids; alternatively, a cleavage enzyme or ribozyme or deoxyribozyme can be configured or engineered to perform off-site cleavage at a non-terminal position of a peptide, e.g., within an amino acid, at n-1, n-2, n-3, n-4, n-5, n-6, n-7, n-8, n-9, n-10, etc., where n is the number of amino acids in the peptide.

[0158] In the case of enzymatic cleavage, additional reagents can be provided to catalyze or induce cleavage. For example, a metalloprotease, aminopeptidase or exopeptidase can promote the cleavage of amino acid or amino acids in the presence of a catalyst (e.g., a metal or metal ion (e.g., cobalt)). Thus, a catalyst can be provided to facilitate the binding of the enzyme to the amino acid or subsequent cleavage of the amino acid from the peptide. In some examples, cleavage can be mediated by an apoenzyme that is inactive in the absence of a metal catalyst of a cofactor, and cleavage can be controlled by the addition of a metal or metal ion.

[0159] Other examples of cleavage stimuli include: light stimuli (e.g., application of UV, X-ray, gamma ray or light of other wavelengths), mechanical stimuli (e.g., sonication, high pressure, electromagnetic energy), thermal stimuli (e.g., application of heat) or chemical stimuli. In some cases, a peptide can contain or be modified to contain a cleavable or labile bond that can be cleaved upon application of an appropriate stimulus, e.g., a disulfide bond (e.g., cleavable upon application of a chemical stimulus such as a reducing agent), an ester bond (e.g., cleavable with a change in pH), a vicinal diol bond (e.g., cleavable with sodium periodate), a Diels-Alder bond (e.g., cleavable upon application of heat), a sulfone bond (e.g., cleavable via a base), a silyl ether bond (e.g., cleavable via an acid), a glycosidic bond (e.g., cleavable via an amylase), a peptide bond (e.g., cleavable via a protease) or a phosphodiester bond (e.g., cleavable via a nuclease (e.g., DNase)).

[0160] Similarly, in some cases, the capture moiety can be cleaved from the peptide or substrate. Cleavage can occur at any useful or convenient step, e.g., after formation of the AALC complex or stacked AALC complexes, after multiple iterations of the workflow to generate multi-stacked AALC complexes, etc. In some cases, cleavage of the capture moiety can occur after formation of the multi-stacked AALC complex, and the cleavage product can be sequenced, e.g., using nanopores.

[0161] Figure 1Schematically shown is an exemplary workflow for generating modified amino acids using an intramolecular amplification process followed by nanopore sequencing. In such an exemplary workflow 100, a peptide 103 and a capture moiety 105 are provided, which are optionally coupled to a substrate 101. The capture moiety 105 can include a first nucleic acid molecule (e.g., a DNA molecule). In process 106, a linker 107 and a polymerizable molecule 109, e.g., a second nucleic acid molecule, are provided. In some cases, the linker 107 is pre-tethered to the polymerizable molecule 109; alternatively, the linker 107 and the polymerizable molecule 109 can be provided separately. In process 106, the linker 107 can be coupled to an amino acid (e.g., NTAA) of the peptide 103 to generate an amino acid-linker complex. In process 110, the amino acid-linker complex can be coupled to the capture moiety 105. The coupling of the amino acid-linker complex to the capture moiety 105 can be mediated by the polymerizable molecule 109. Optionally, the amino acid-linker complex and the capture moiety can be covalently linked together using chemical (e.g., click chemistry) or enzymatic (e.g., ligase) methods (e.g., the polymerizable molecule 109 can be covalently linked to the capture moiety 105). Alternatively, or in addition, the polymerizable molecule 109 can include a first sequence (not shown) that is complementary and hybridizable to a second sequence of the capture moiety 105, or the polymerizable molecule 109 can be linked to the capture moiety 105 via a splint or bridge molecule that can include a sequence complementary to the first sequence of the polymerizable molecule 109 and the second sequence of the capture moiety 105 (not shown). In process 112, the amino acid can be removed or cleaved from the peptide 103 to generate an amino acid-linker-capture (AALC) complex 113, including a modified amino acid (e.g., NTAA modified due to cleavage from the peptide), the linker 107, the polymerizable molecule 109, and the capture moiety 105. Processes 106, 110, and 112 can be repeated any number of times ("rounds"), using additional linkers 107 and polymerizable molecules 109 and tethering the additional polymerizable molecules together (e.g., tethering the additional polymerizable molecules to the polymerizable molecule of the AALC complex 113). Multiple rounds can be continued until all or part of the amino acids in the peptide 103 are removed from the peptide and tethered together. For example, a modified amino acid derivative (e.g., a complex 115 containing multiple modified amino acids) can be generated by n rounds (e.g., iterations of processes 106, 110, and 112) of the workflow 100.

[0162] As described elsewhere herein, modified amino acids can include amino acids having one or more modifications. For example, with respect to Figure 1, a modified amino acid can be used to refer to an amino acid-linker complex (e.g., produced by process 106) or a portion thereof (e.g., only the amino acid of the amino acid-linker complex), an amino acid-linker-polymerizable molecule complex, an amino acid-linker-polymerizable molecule-capture moiety complex (e.g., produced by process 110) or a portion thereof, a cleaved amino acid of AALC complex 113 or a portion thereof, or an amino acid of complex 115 of an AALC complex (stacked AALC complex). It should be understood that modified amino acids can include Figure 1 other additional modifications not depicted in Figure 1 , such as post-translational modifications or chemical modifications (e.g., protecting groups) described elsewhere herein. In some cases, modified amino acids include derivatized amino acids. For example, the linker can contain a PITC moiety as an amino acid-reactive group, and after conjugating PITC with an amino acid of a peptide (e.g., NTAA) under mild basic conditions, a phenylthiocarbamoyl (PTC) derivative of the amino acid is generated. The PTC-derivatized amino acid can be treated with an acid (e.g., TFA or a Lewis acid) to generate a cleaved cyclic 2-anilino-5(4)-thiazolinone (ATZ) -derivatized amino acid, leaving a new N-terminus on the remaining peptide. The ATZ-derivatized amino acid can be converted to a phenylthiohydantoin (PTH) derivative or a PTC derivative.

[0163] A “derivative” of a modified amino acid generally can refer to a molecule derived from the modified amino acid. The derivative can be a product of a reaction (e.g., chemical, enzymatic reaction) or interaction of the modified amino acid with another molecule. For example, referring to Figure 1 , a modified amino acid can be an amino acid of an amino acid-linker complex (e.g., produced by process 106), and a derivative of the modified amino acid can refer to all or part of the following: (i) an amino acid-linker complex conjugated to a capture moiety (e.g., produced by process 110), (ii) AALC complex 113, or (iii) complex 115 of an AALC complex (stacked AALC complex). Optionally, the modified amino acid can be further processed to obtain “its derivative”. For example, the modified amino acid can be further chemically or enzymatically treated, the capture moiety cleaved, extended or amplified, removed from the substrate, or physically processed (e.g., mechanical shearing or fragmentation) to obtain a derivative of the amino acid.

[0164] Substrates: One or more molecules described herein can be coupled to a substrate. The one or more molecules coupled to the substrate can be of the same type or different types of molecules. For example, a capture moiety (e.g., a nucleic acid molecule) and a peptide can be coupled to the substrate by covalent or non-covalent interactions. The capture moiety and the peptide can be coupled to the substrate using any suitable chemical method, such as click chemistry moieties (e.g., alkyne-azide coupling), photoreactive groups (e.g., benzophenone), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) (e.g., for coupling amino oligonucleotides or peptides), N-hydroxysulfosuccinimide (NHS), sulfo-NHS, or NHS-esters (e.g., for coupling thiol oligonucleotides), maleimide, thiol, biotin-streptavidin interaction, cystamine, glutaraldehyde, formaldehyde, SMCC, sulfo-SMCC, silanes (e.g., aminosilanes), combinations thereof, etc. In some cases, the substrate can be functionalized to include coupling chemical methods for coupling peptides or capture moieties. In one non-limiting example, the substrate (e.g., a bead or a surface) can include an alkyne, such as dibenzocyclooctyne (DBCO), which can be configured to react with amines (e.g., DBCO-alcohol, DBCO-Boc), carboxyl groups, or carbonyl groups (e.g., DBCO, DBCO-silane), thiols, etc. Azide-functionalized nucleic acids or proteins can react with DBCO to link the nucleic acid or protein to the DBCO substrate. In other examples, linkers such as bifunctional linkers can be used to attach molecules to the substrate; such bifunctional linkers can include the same reactive moieties at both ends, or different moieties at each end (e.g., heterobifunctional linkers).

[0165] Referring again Figure 1 , complex 115 of the AALC complex (stacked AALC complex) can be further processed to prepare complex 115 for further characterization, such as by sequencing. For example, in the case where the capture moiety 105 is tethered to the substrate 101, complex 115 can be removed from the substrate. For example, the capture moiety 105 can include a releasable or cleavable moiety, such as a restriction site recognized by a restriction enzyme, a chemically cleavable linkage, such as a disulfide bond, a photosensitive linkage cleavable upon application of light stimulation, etc., as described elsewhere herein. Alternatively, or in addition, the capture moiety 105 can include a primer binding site such that the stacking of polymerizable molecules (e.g., nucleic acid molecules) can be replicated or amplified, e.g., using a primer extension reaction, for downstream analysis or sequencing. Additional operations can be performed, such as purification or enrichment, cleaning, nucleic acid reactions (e.g., ligation, extension, amplification, tagging, restriction enzyme cleavage), fragmentation, barcoding, addition of adapters (e.g., sequencing adapters, read sequences, etc.), enzymatic treatment, etc.

[0166] Nanopore sequencing: Nanopore sequencing can be performed on modified amino acids or their derivatives to determine the identity of the amino acid (e.g., one of the proteinogenic naturally occurring amino acids) and optionally the identity of the polymerizable molecule. Nanopore sequencing can be carried out using commercially available nanopore systems, such as Oxford Nanopore Technologies, Genia Technologies, NobleGen, or Quantum Biosystem. Nanopore sequencing can be performed to determine the identity of the different components of the modified amino acid; for example, nanopore sequencing can be carried out on a modified amino acid that includes a derivatized amino acid (e.g., a thiocyanate-conjugated amino acid or a thiocarbamoyl, thiazolinone, or thiohydantoin derivative) coupled to a polymerizable molecule (e.g., a nucleic acid molecule), which can output the identity of which amino acid type the modified amino acid includes or is derived from (e.g., which one of the 20 proteinogenic amino acids or post-translationally modified amino acids), and optionally, the identity of the individual monomers of the polymerizable molecule (e.g., the nucleic acid sequence of the nucleic acid molecule). In the case where the polymerizable molecule includes a nucleic acid molecule encoding additional information (e.g., including a barcode sequence, UMI, circular information, spatial information, etc.), sequencing of the derivatized amino acid and the nucleic acid molecule can yield multiplexed information.

[0167] Multiplexed sequencing: Advantageously, the use of nanopore sequencing to analyze peptides using the methods provided herein can simplify the process of generating multiplexed data. For example, the methods provided herein can allow for the identification of amino acid residues and polymerizable molecules (e.g., identification of individual monomers, nucleic acid sequences of nucleic acid molecules). As described herein, modified amino acids can include polymerizable molecules (e.g., nucleic acid molecules) and amino acids or their derivatives. Polymerizable molecules can include multiple types of information. For example, a polymerizable molecule (e.g., a nucleic acid molecule) can include sequences encoding cyclic or other temporal information (as described above) as well as spatial information. In one such example, an array of peptides and capture moieties can be provided on a substrate. The array can include multiple individually addressable units, where each (or subset) of the individually addressable units of the array includes a peptide to be sequenced and a capture moiety. Linkers can be provided on the array and can be attached to the amino acids of the peptide. Before, during, or after providing the linker, multiple polymerizable molecules can be provided. The multiple polymerizable molecules can contain spatial information (e.g., spatial barcode sequences) that uniquely identify the individually addressable units and thus identify the location of the array. Alternatively, or in addition, the polymerizable molecule or the capture moiety can contain information about the peptide, e.g., a barcode identifying the peptide, the partition, sample, cell population, etc. from which the peptide is derived. The polymerizable molecule or the capture moiety can also additionally contain temporal information (e.g., a cyclic barcode indicating the round or iteration in which the polymerizable molecule or the capture moiety was provided). Subsequent sequencing can be used to reveal spatial or temporal information (e.g., the starting position of the peptide or amino acid in the array, the cycle or time in which the polymerizable molecule or the capture moiety was provided, etc.). Alternatively, or in addition, the capture moiety can contain multiplexing information (e.g., a barcode sequence, e.g., for identifying the sample or partition from which the peptide or the peptide is derived, spatial information, temporal information, UMI, etc.).

[0168] Similarly, a binder can be used, which can add additional multiplexing analysis methods for peptide sequencing. A binder conjugated to a modified amino acid can be used to modulate the translocation of the modified amino acid through the nanopore. The binder can contain additional information that can be read out using nanopore sequencing. For example, different binders that recognize specific amino acid residues (or modified amino acids or amino acid-linker complexes) can be used; different binders, alone or in combination with the modified amino acids to which they are conjugated, can each be associated with a unique current signature when translocating through the nanopore and can thus be used to identify specific residues. In some cases, the binder can further include additional coding information, such as by conjugate barcode molecules or detectable tags. For example, the binder can have a barcode nucleic acid molecule conjugated to it, which can identify the binder or the binding partner of the binder (such as an amino acid residue). Alternatively or in addition, the binder can contain spatial or temporal information, as described above. Thus, when a modified amino acid containing a binder associated with it translocates through the nanopore, multiple sets of information can be obtained, such as spatial, temporal, coded barcode information from the binder, the amino acid or modified amino acid, the polymerizable molecule, the capture moiety, or a combination thereof.

[0169] Advantageously, the methods provided herein can generate multiplexed information in a simplified workflow, thereby eliminating the need for multiple analytical instruments. Thus, acquisition of multiplexed data can occur substantially simultaneously. For example, using nanopore sequencing, the identity of an amino acid (or modified amino acid) and the identity of the polymerizable molecule conjugated to it can be obtained in at most about 5 minutes, about 1 minute, about 50 seconds, about 40 seconds, about 30 seconds, about 20 seconds, about 10 seconds, about 1 second, about 900 milliseconds, about 800 milliseconds, about 700 milliseconds, about 600 milliseconds, about 500 milliseconds, about 400 milliseconds, about 300 milliseconds, about 200 milliseconds, about 100 milliseconds, about 900 microseconds, about 800 microseconds, about 700 microseconds, about 600 microseconds, about 500 microseconds, about 400 microseconds, about 300 microseconds, about 200 microseconds, about 100 microseconds, about 900 nanoseconds, about 800 nanoseconds, about 700 nanoseconds, about 600 nanoseconds, about 500 nanoseconds, about 400 nanoseconds, about 300 nanoseconds, about 200 nanoseconds, about 100 nanoseconds, or less.

[0170] Arrays: The methods and systems provided herein can include the use of arrays, such as for large-scale parallel sample processing or sequencing. In one example, a substrate can include an array of individually addressable units that contain multiple peptide analytes. In some cases, a subset of the individually addressable units includes a single peptide analyte. The individually addressable units can additionally include a single or multiple capture moieties. In some cases, a subset of the individually addressable units that contain a single peptide each includes at least one capture moiety. The arrays can be patterned (e.g., the individually addressable units are arranged in a pattern), or they can be random (e.g., the individually addressable units are randomly distributed on the substrate).

[0171] An array can contain any useful number of individually addressable units. For example, an array can have at least 5, at least 10, at least 100, at least 1,000, at least 10,000, at least 100,000, at least 1,000,000, at least 10,000,000, at least 100,000,000, at least 1,000,000,000 or more individually addressable units. Alternatively, an array can have at most about 1,000,000,000, at most about 100,000,000, at most about 10,000,000, at most about 1,000,000, at most about 100,000, at most about 10,000, at most about 1,000, at most about 100, at most about 10, or at most about 5 individually addressable units. An array can have a number of individually addressable units, e.g., from about 1,000 to about 100,000, or from about 50,000 to about 1,000,000 individually addressable units.

[0172] Binding agents: In some embodiments, the methods, compositions, systems, and kits provided herein can include the use of binding agents. A binding agent can be or include a protein or peptide (e.g., an antibody, an antibody fragment, a nanobody), a nucleic acid molecule (e.g., an aptamer), a polymer, an inorganic compound, a small molecule, a derivative (e.g., an engineered variant), or a combination thereof. A binding agent can bind to a modified amino acid or a moiety thereof. The use of a binding agent can be used to identify a single amino acid (or a modified amino acid) of a peptide or to modulate the translocation of a modified amino acid through a nanopore or a nanogap. A binding agent can include a recognition site that specifically recognizes an amino acid, a modified amino acid, or a derivatized (and optionally modified) amino acid. For example, a binding agent can be configured to recognize a part of a modified amino acid, such as a specific amino acid residue, a residue-linker complex, or a derivatized amino acid (e.g., a thiocarbonyl-derived residue, a thiazolone-derived residue, a thiohydantoin-derived residue, etc.), or a part of a modified amino acid. In some cases, a binding agent can be derived from or designed from a naturally occurring enzyme, such as an aminopeptidase or a tRNA synthetase.

[0173] In some cases, the binder can be provided as part of an intramolecular extension process; for example, the binder can specifically recognize and bind a modified amino acid (e.g., an AALC complex, an amino acid conjugated to a polymerizable molecule) or a part thereof. In some cases, the binder can recognize and bind a part of a modified amino acid, e.g., an amino acid-linker conjugate or a derivative thereof (e.g., isothiocyanate-amino acid or its thiocarbamoyl, thiazolone, or thiohydantoin derivative). The binder can comprise a detectable moiety (e.g., a fluorophore, a barcode molecule, a radioisotope, or other label) that can facilitate the detection and identification of individual amino acids. Alternatively, or in addition, the binder can be recognized by an additional binder (e.g., a secondary antibody) comprising a detectable moiety; the binding of the additional binder to the binder can represent the presence of the modified amino acid.

[0174] Figure 2Another exemplary workflow for in-peptide molecule amplification is schematically shown, including the use of a binder. In workflow 200, a peptide 203 and a capture moiety 205 are provided, which is optionally conjugated to a substrate 201. The capture moiety 205 can include a first nucleic acid molecule (e.g., DNA). In process 206, a linker 207 and a polymerizable molecule 209, such as a second nucleic acid molecule, are provided. In some cases, the linker 207 is pre-tethered to the polymerizable molecule 209; alternatively, the linker 207 and the polymerizable molecule 209 can be provided separately. In process 206, the linker 207 can be conjugated to an amino acid (e.g., NTAA) of the peptide 203 to generate an amino acid-linker complex. In process 210, the amino acid-linker complex can be conjugated to the capture moiety 205. The conjugation of the amino acid-linker complex to the capture moiety 205 can be mediated by the polymerizable molecule 209. Optionally, the amino acid-linker complex and the capture moiety can be covalently linked together using chemical methods (e.g., click chemistry) or enzymatic methods (e.g., ligase) (e.g., the polymerizable molecule 209 can be covalently linked to the capture moiety 205). Alternatively, or in addition, the polymerizable molecule 209 can include a first sequence (not shown) that is complementary and hybridizable to a second sequence of the capture moiety 205, or the polymerizable molecule 209 can be linked to the capture moiety 205 via a splint or bridge molecule that can include a sequence (not shown) complementary to the first sequence of the polymerizable molecule 209 and the second sequence of the capture moiety 205. In process 212, the amino acid can be removed from the peptide 203 to generate an amino acid-linker-capture (AALC) complex 213, which includes a modified amino acid (e.g., NTAA modified due to cleavage from the peptide), the linker 207, the polymerizable molecule 209, and the capture moiety 205. In process 214, a binder 217 (e.g., an antibody, a binding protein, etc.) can be contacted with the AALC complex 213. The binder can be configured to recognize all or part of the AALC complex 213. For example, the binder can recognize the residue of the modified amino acid, or the binder can recognize all or part of the residue and the linker 207. In one example, the linker can include a PITC moiety, and the binder can recognize PITC-amino acid or its derivatives. For example, phenylthiocarbamyl, thiazolone, or phenylthiohydantoin. In some cases, the binder 217 can include a detectable moiety (not shown) or can be contacted with an additional binder (e.g., a secondary antibody) that optionally includes a detectable moiety (not shown).

[0175] Additional linker 207 and polymerizable molecule 209 (optionally containing cycle / round information) can be used and additional polymerizable molecules can be tethered together (e.g., tethering additional polymerizable molecules to the polymerizable molecules of the AALC complex 113), and processes 206, 210, 212, and 214 can be iterated and repeated any number of times (“rounds”). Multiple rounds can be continued until all or some of the amino acids in peptide 203 are removed from the peptide and tethered together. For example, a modified amino acid derivative resulting from n rounds of the workflow 200 (e.g., iterations of processes 206, 210, 212, and optionally 214), such as complex 215 containing multiple modified amino acids.

[0176] Intramolecular amplification can be performed in solution or using a substrate-free method. Figure 3Another exemplary workflow for generating modified amino acids using an intramolecular amplification process in solution, followed by nanopore sequencing, is schematically shown. In such an exemplary workflow 300, a peptide 303 and a capture moiety 305 are provided. The capture moiety 305 can include a first nucleic acid molecule (e.g., a DNA molecule) and can include identification information of the peptide 303, e.g., a peptide recognition barcode sequence. The capture moiety 305 can also include a releasable or cleavable moiety. In process 306, a linker 307 and a polymerizable molecule 309, e.g., a second nucleic acid molecule, are provided. In some cases, the linker 307 is pre-tethered to the polymerizable molecule 309; alternatively, the linker 307 and the polymerizable molecule 309 can be provided separately. The polymerizable molecule 309 can include identification time information, e.g., a cycle or round providing such information. In process 306, the linker 307 can be coupled to an amino acid (e.g., NTAA) of the peptide 303 to generate an amino acid-linker complex. In process 310, the amino acid-linker complex can be coupled to the capture moiety 305. The coupling of the amino acid-linker complex to the capture moiety 305 can be mediated by the polymerizable molecule 309 and optionally an additional polymerizable molecule 311. Optionally, the amino acid-linker complex and the capture moiety can be covalently linked together (e.g., by ligation). Alternatively, or in addition, the polymerizable molecule 309 can contain a first sequence (not shown) complementary and hybridizable to a second sequence of the capture moiety 305, or the polymerizable molecule 309 can be linked to the capture moiety 305 via a splint or bridge molecule that can contain a sequence complementary to the first sequence of the polymerizable molecule 309 and the second sequence of the capture moiety 305 (not shown). In process 312, the amino acid can be removed or cleaved from the peptide 312 to generate an amino acid-linker-capture (AALC) complex 313 that includes a modified amino acid (e.g., NTAA modified due to cleavage from the peptide), the linker 307, the polymerizable molecule 309, and the capture moiety 305. Processes 306, 310, and 312 can be repeated any number of times ("rounds"), using additional linkers 307 and polymerizable molecules 309 and tethering the additional polymerizable molecules together (e.g., tethering the additional polymerizable molecules to the polymerizable molecule of the AALC complex 313). Multiple rounds can be continued until all or part of the amino acids in the peptide 303 are removed from the peptide and tethered together. For example, a modified amino acid derivative (e.g., a complex 315 containing stacked AALC complexes that includes multiple modified amino acids) can be produced by n rounds (e.g., iterations of processes 306, 310, and 312) of the workflow 300. After any useful number of rounds, the complete stacked AALC complex can be cleaved from or at the capture moiety 305, e.g., using the cleavable moiety. The cleavage product can then be sequenced using a nanopore or nanogap method.

[0177] In some cases, the polymerizable molecule 309 includes time information that provides for its cycling; thus, the time information can be used for quality control. For example, if a certain number of cycles are missing, it can be inferred that an amino acid is missing or absent in the peptide, that the cleavage of the amino acid did not occur, or that other errors exist.

[0178] It should be understood that the order of operations and processes provided herein is listed for illustrative purposes only, and that any operation and process can be performed at any convenient or useful step or time. For example, the polymerizable molecule can be provided pre-coupled to a linker (e.g., as shown in process 106 of Figure 1 process 106, Figure 2 process 206, and Figure 3 process 306), or the polymerizable molecule can be provided before or after the linker is provided. Similarly, the coupling of the polymerizable molecule to the capture moiety can occur before, during, or after the coupling of the linker to the amino acid. In another example, the removal of the amino acid from the peptide (e.g., cleavage) can occur before, during, or after the coupling of the polymerizable molecule to the capture moiety. In another example, in the case of performing binder and intramolecular amplification, the binder can be introduced before, during, or after the intramolecular process. For example, the operations of workflows 100, 200, and 300 can be performed, and the stacked AALC complexes (115, 215, and 315) can be contacted with a plurality of binders (e.g., 10, 20, or more different binders, each binder specific for a different amino acid or modified amino acid) that are specific for individual or oligomeric (e.g., dipeptide or tripeptide) of different modified amino acids. The plurality of binders can include a detectable label that can be used for amino acid recognition (e.g., using single molecule imaging); alternatively, or in addition, the complex of the AALC complex and the binder can be introduced into a nanopore or nanogap for sequencing.

[0179] Additional operations of the methods described herein are also contemplated. For example, reference Figures 1-3, in some cases, the AALC complex 113, 213, or 313 or a portion of the AALC complex can be cleaved or removed from the substrate or peptide (e.g., by cleavage, enzymatic digestion, chemical dissociation) prior to iteration of workflow 100, 200, or 300. In such an example, the cleaved molecule (e.g., including modified amino acids and optional polymerizable molecules) can be translocated through the nanopore for protein sequencing. In another example, additional cleavage or dissociation reactions can be performed to separate the amino acids from the polymerizable molecules. In this case, the separated polymerizable molecules or amino acids can be translocated through the nanopore. Other useful operations, methods, systems, and compositions for handling or using peptides, linkers, polymerizable molecules, etc. can also be found in PCT / US2023 / 017954, which is hereby incorporated by reference in its entirety.

[0180] Any of the operations described herein can occur under any useful physical conditions. For example, one or more of the operations described herein can occur at elevated or reduced temperatures, either of which can contribute to reaction rate, reaction completion, translocation rate or dwell time of molecules through the nanopore or nanogap, etc. In some cases, nanopore or nanogap sequencing systems can be performed on ice or at reduced temperatures, which can beneficially increase the signal-to-noise ratio of the measured current signal, regulate the translocation rate of molecules through the nanopore or nanogap, or otherwise improve readout or detection.

[0181] Detection: Alternatively or in addition to nanopore or nanogap sequencing, additional methods and devices for sequencing peptides and / or polymerizable molecules can be used. For example, the methods provided herein can utilize one or more imaging systems to identify molecules (e.g., modified amino acids or polymerizable molecules). Examples of optical imaging systems and methods include, but are not limited to: epifluorescence microscopy, confocal microscopy, total internal reflection fluorescence (TIRF) microscopy, expansion microscopy, two-photon microscopy, integrated correlation microscopy, stimulated emission depletion (STED) microscopy, stochastic optical reconstruction microscopy (STORM), reversible saturable optical linear fluorescence transitions (RESOLFT), spatially modulated illumination, spectral precision distance microscopy (SPDM), photoactivated localization microscopy (PALM), fluorescence PALM (FPALM), structured illumination microscopy (SIM), saturated SIM (SSIM), etc.

[0182] In some cases, imaging can be used to identify modified amino acids or polymerizable molecules. For example, refer to Figure 2, a binding agent (e.g., an antibody) can carry a fluorophore or other optically detectable label, and the binding agent can selectively bind to a specific residue of the modified amino acid (e.g., in process 217). The fluorophore can identify the binding agent such that the presence of the fluorophore indicates the presence of the binding agent (and the amino acid residue to which the binding agent binds). Alternatively, a secondary binding agent (e.g., a secondary antibody) carrying a fluorophore or other detectable agent can bind to the binding agent (e.g., a primary antibody) to indicate the presence of the binding agent (and the amino acid residue to which the primary antibody binds). Similarly, for a polymerizable molecule (e.g., a nucleic acid molecule), any suitable imaging technique (e.g., fluorescence in situ hybridization or synthetic sequencing) can be used to determine the sequence of the nucleic acid molecule.

[0183] Alternatively, or in addition, other proteomics tools can be used to identify the modified amino acid and the polymerizable molecule. For example, mass spectrometry, immunochemistry (e.g., by ELISA, fluorescence imaging), or other analytical techniques can be used to analyze the modified amino acid (e.g., an amino acid that has contacted a PITC linker coupled to a DNA molecule). In some cases, the modified amino acid can be further processed prior to analysis. For example, such further processing can include separating, enriching, or purifying the modified amino acid from the substrate, or adding a label or other moiety. In some cases, separation techniques can be performed on the modified amino acid prior to analysis, such as chromatography (e.g., liquid chromatography, HPLC, nanoLC), electrophoretic separation (e.g., electrophoresis, isoelectric focusing, electroosmotic flow), filtration, evaporation, distillation, or other separation techniques.

[0184] In another aspect of the present disclosure, a method of analyzing an analyte is disclosed, comprising: (a) providing a polymerizable molecule (e.g., a nucleic acid molecule) conjugated to the analyte, (b) sequencing the polymerizable molecule (e.g., the nucleic acid molecule), and (c) identifying the analyte, wherein (b) and (c) are performed substantially simultaneously.

[0185] The analyte can include a non-nucleic acid biomolecule (e.g., a protein or peptide, lipid, carbohydrate, a combination thereof) or a component thereof. In some cases, the analyte is a peptide comprising at least two amino acids. In some cases, the analyte includes a modified amino acid as described elsewhere herein (e.g., a modified amino acid generated from a peptide by intramolecular amplification and using a linker). In some cases, the analyte includes a modified amino acid, a linker, and a polymerizable molecule (e.g., a nucleic acid molecule) as described elsewhere herein.

[0186] As described herein, an analysis method can include using a nanopore or a nanogap for sequencing or identification. Nanopore or nanogap sequencing methods can be used to generate multiplexed data, such as (i) sequencing of polymerizable molecules (e.g., sequencing of nucleic acid molecules to generate sequencing reads) and (ii) identification of analytes (e.g., identification of amino acid residues). Generation of such multiplexed data can occur substantially simultaneously, such as within about 5 minutes, about 1 minute, about 1 second, about 1 millisecond, about 1 microsecond, about 1 picosecond, or less. Alternatively, or in addition, sequencing and identification can not occur substantially simultaneously, but signal generation from polymerizable molecules and amino acids can occur substantially simultaneously. For example, a modified amino acid or its derivative (e.g., an AALC complex, a stacked AALC complex, or a portion thereof) can translocate through a nanopore. When the modified amino acid or its derivative translocates through the nanopore, one or more signals (e.g., current signals) generated by the polymerizable molecules and amino acids can be generated and collected. Then, computational methods can be used to deconvolute such one or more signals to determine the identity of the polymerizable molecule (e.g., nucleic acid sequence) and the amino acid type of the modified amino acid or its derivative. Advantageously, using nanopore or nanogap sequencing to generate multiplexed data can obviate the need for multiple analytical techniques or instruments.

[0187] Systems, kits, compositions: Also provided herein are systems, kits, and compositions for characterizing an analyte (e.g., a peptide). The systems, kits, and compositions provided herein can be used to implement any of the described methods or can be provided as a supplement to the described methods.

[0188] In one aspect, a kit of the invention can include a substrate, a capture moiety, a linker, a polymerizable molecule, a peptide-conjugating reagent, an enzyme (e.g., a polymerase or ligase, a cleavage enzyme, a restriction enzyme, a nicking enzyme, an exonuclease, a repair enzyme (e.g., uracil DNA glycosylase)), a detection or labeling agent, or any combination thereof. The kit can include buffers, reagents, binding agents, catalysts, or other chemicals or biomolecules (e.g., enzymes) necessary to perform chemical or enzymatic reactions. The kits of the present disclosure can also include instructions for using the components of the kit or implementing any of the methods and procedures described herein. For example, the kit can include instructions for conjugating a peptide to a substrate using a peptide-conjugation reaction. The kit can contain instructions for conjugating the capture moiety to the substrate, or alternatively, the substrate can contain a capture moiety conjugated thereto. Similarly, the kit can contain instructions for performing an in-molecule amplification described herein, e.g., generating a modified amino acid, an amino acid-linker complex, an AALC complex, etc. that contains a polymerizable molecule (e.g., DNA) conjugated thereto.

[0189] In another aspect, the present disclosure provides compositions that can be used to characterize an analyte (e.g., a peptide). The compositions can include a linker covalently attached to a polymerizable molecule (e.g., a nucleic acid molecule), which can be used, for example, for intramolecular amplification of a peptide for peptide sequencing. In one example, the composition can include (A) a linker (including (i) a first portion that can couple to an amino acid (e.g., the CTAA or NTAA of a peptide), (ii) a second portion that can couple to a nucleic acid molecule (e.g., DNA), and optionally, (iii) a releasable or cleavable portion, which can be the same or different from (ii), and optionally, (iv) a spacer portion) and (B) a nucleic acid molecule. In another example, the composition can comprise a linker covalently coupled to a nucleic acid molecule; such a linker can include a portion that can couple and optionally cleave an amino acid (e.g., the CTAA or NTAA of a peptide), and the covalently coupled nucleic acid molecule can be configured to tether to another nucleic acid molecule (e.g., a capture portion), which in some cases can be attached to a substrate.

[0190] Figure 4 Schematically shown are example compositions described herein. Figure 4 Partition A shows a linker 403 that can be coupled to a polymerizable molecule 401 (e.g., a DNA molecule). The polymerizable molecule 401 can comprise a first reactive group, such as an azide moiety (represented as N=N + =N - ), which can be attached to the polymerizable molecule 401 at any useful position (e.g., for a DNA molecule, at the 5'-end, 3'-end, or an internal position of the DNA molecule). The linker 403 can comprise (i) an amino acid-reactive portion, such as thiocyanate (e.g., as shown N=C=S) or PITC, and (ii) a second reactive group (e.g., an alkyne) capable of reacting with the first reactive group (e.g., an azide) of the polymerizable molecule 401. Figure 4 Partition B shows an example of a polymerizable molecule covalently coupled to a linker. The first reactive group (e.g., an azide) of the polymerizable molecule can react with the second reactive group (e.g., an alkyne) of the linker, thereby generating a covalently linked structure comprising the polymerizable molecule and the linker, which may be capable of reacting and tethering to an amino acid. As described herein, the amino acid-reactive group (e.g., ITC, PITC) may also be capable of cleaving an amino acid from a peptide. In some cases, the first reactive group is attached to the polymerizable molecule via an additional linker. For example, the additional linker can include a nucleic acid linker that includes a modified nucleobase or sugar backbone that can be incorporated into a nucleic acid molecule, such as a nucleoside, nucleoside monophosphate, nucleoside diphosphate, or nucleoside triphosphate. The additional linker can also include a first reactive group, such as a click chemistry moiety. In some examples, the additional linker includes ethynyl deoxyuridine or octadiynyl deoxyuridine.

[0191] Systems, Compositions, and Kits: Also provided herein are systems, compositions, and kits for processing peptides or for generating or processing modified amino acids. The systems of the present disclosure can include a sequencing instrument configured to receive a modified amino acid or a derivative thereof described herein and provide sequencing reads of the modified amino acid or a derivative thereof. Alternatively, or in addition, the systems of the present disclosure can be configured to process, prepare, or sequence modified amino acids. The system can be configured to provide a peptide, a capture moiety, and a linker comprising a polymerizable molecule; couple the linker to an amino acid of the peptide; cleave an amino acid from the peptide; and optionally, iterate one or more operations or processes. Thus, the systems of the present disclosure can include any useful device or tool, including but not limited to mixers, liquid handlers, vortexers, centrifuges, heating or cooling elements, robotic stages, microfluidic chambers or devices, and fluid controls.

[0192] The systems of the present disclosure can include a nanopore or nanochannel sequencing system configured to sequence or analyze modified amino acids. The modified amino acids can include or be conjugated to a polymerizable molecule as described elsewhere herein. Thus, the system can be configured to output multiplexed data regarding the modified amino acid (e.g., which of the 20 proteinogenic amino acid residues the modified amino acid is or is derived from) and the polymerizable molecule (e.g., nucleic acid sequence). The systems of the present disclosure can include a substrate, a capture moiety, a linker, a polymerizable molecule, a peptide conjugating reagent, an enzyme (e.g., a polymerase or ligase, a cleavage enzyme, a restriction enzyme, a nickase, an exonuclease, a repair enzyme (e.g., uracil DNA glycosylase), a detection or labeling agent, a buffer, a reagent, a binding agent, a catalyst, or other chemicals or biomolecules (e.g., enzymes) required to perform a chemical or enzymatic reaction, or any combination thereof. The system can also include one or more detection (e.g., imaging or mass spectrometry) systems, separation systems (e.g., HPLC), or other analytical instruments.

[0193] The compositions of the present disclosure can include any useful article or reagent for processing or sequencing peptides or for generating modified amino acids or derivatives thereof. The compositions can include a capture moiety configured to couple to a polymerizable molecule. The compositions can include a linker (e.g., coupled to an amino acid) that can include a polymerizable molecule, a cleavage agent, a ligating agent (e.g., a ligase), a capture moiety, a reagent for performing a reaction, or a combination thereof. In some cases, the compositions include a linker that includes (e.g., is covalently attached to) a nucleic acid barcode molecule, wherein the linker includes an amino acid-reactive group. The nucleic acid barcode molecule can include any useful barcode sequence, such as a spatial, temporal, peptide recognition, partition recognition, sample recognition sequence, UMI, or other functional sequence.

[0194] The kits of the present disclosure may comprise any useful reagents for processing, analyzing, or sequencing peptides. The kits may comprise reagents for providing peptides and capture moieties, reagents for coupling peptides and capture moieties to substrates, reagents for cleaving amino acids, reagents for coupling peptides to linkers comprising amino acid reactive groups and additional reactive groups or polymerizable molecules, reagents for coupling capture moieties to polymerizable molecules, reagents for providing binders, cleavage reagents, stabilizing reagents, wash buffers, or combinations thereof. The kits may further comprise reagents for removing or deconjugating amino acids from peptides, or reagents for removing or deconjugating AALC complexes from substrates or capture moieties. In some cases, the kits may further comprise reagents for removing or deconjugating AALC complexes or stacked AALC complexes from substrates, or the kits may further comprise reagents for performing nucleic acid extension reactions. The kits may include any relevant reagents, for example, buffers, detergents, chelating agents, cofactors, enzymes, ribozymes or deoxyribozymes, acids, bases, salts, metal ions, primers, nucleic acid molecules, nucleotides, proteins, polynucleotides, binders (e.g., antibodies, aptamers, nanobodies, antibody fragments), lipids, carbohydrates, ribozymes, riboswitches, probes, fluorophores, oxidizing agents, reducing agents, nuclease or protease inhibitors, dyes, organic molecules, inorganic molecules, emulsifiers, surfactants, stabilizers, polymers, water, small molecules, therapeutic agents, radioactive substances, preservatives, or other useful reagents. The kits of the present disclosure may also provide instructions for use of the kit contents.

[0195] Substrate conjugation

[0196] The present disclosure provides methods for coupling a molecule (e.g., a polymeric analyte, such as a biomolecule such as a nucleic acid molecule, a peptide, a lipid, a carbohydrate, etc.) to a substrate. The substrate can be functionalized to allow for covalent or non-covalent coupling of the molecule to the substrate. The substrate can comprise any useful functional moiety, e.g., a reactive moiety that can be coupled or conjugated to the molecule or another reactive moiety. In non-limiting examples, the reactive moiety can include click chemistry moieties such as azides, alkynes, nitrones, alkenes (e.g., strained alkenes), tetrazines, methyltetrazines, triazoles, tetrazoles, phosphites, phosphines, etc. The click chemistry moieties can be reactive in a copper-catalyzed Huisgen cycloaddition or a 1,3-dipolar cycloaddition between an azide and a terminal alkyne, a Diels-Alder reaction (e.g., a cycloaddition between a diene and a dienophile), or a nucleophilic substitution reaction in which one of the reactive species is an epoxide or an aziridine. The molecule to be coupled to the substrate can comprise a click chemistry moiety that is complementary to the click chemistry moiety of the substrate; for example, the substrate can comprise an alkyne moiety and the molecule to be coupled can comprise an azide moiety that can react with the alkyne moiety of the substrate to produce a covalent linkage. In one such example, the substrate can comprise a dibenzocyclooctyne (DBCO) moiety and a molecule comprising an azide (e.g., azide-DNA, azide-polymer, azide-peptide) can react and conjugate to the dibenzocyclooctyne moiety.

[0197] Alternatively or additionally, the reactive moiety can include a photoreactive moiety that can be activated when exposed to a light stimulus (e.g., light such as UV or visible light). Examples of photoreactive moieties include aryl (phenyl) azides (e.g., azidobenzene, o-hydroxyazidobenzene, m-hydroxyazidobenzene, tetrafluorophenyl azide, o-nitroazidobenzene, m-nitroazidobenzene), diazirines, azido-methyl-coumarin, benzophenone, anthraquinone, diazo compounds, diazirines, psoralens, 3-cyano-vinylcarbazole phosphoramidite (CNVK), and their analogs or derivatives.

[0198] The reactive moiety may contain carboxyl-reactive crosslinker groups such as diazo compounds such as diazomethane and diazoacetyl, carbonyldiimidazole, carbodiimides (e.g., 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC)), dicyclohexylcarbodiimide (DCC)), or amine-reactive groups (e.g., N-hydroxysulfosuccinimide (NHS), sulfo-NHS, or NHS-esters). The reactive groups may include crosslinkers, which may contain NHS groups, EDC groups, maleimides (e.g., for coupling to Michael acceptors), thiols, cystamine, aldehydes, succinimidyl groups, epoxides, acrylates. Examples of crosslinkers include, for example, NHS (N-hydroxysuccinimide); sulfo-NHS (N-hydroxysulfosuccinimide); EDC (1-ethyl-3-[3-dimethylaminopropyl]); carbodiimide hydrochloride; SMCC (4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid succinimidyl ester); sulfo-SMCC; DSS (disuccinimidyl suberate); DSG (disuccinimidyl glutarate); DFDNB (1,5-difluoro-2,4-dinitrobenzene); BS3 (bis(sulfosuccinimidyl)suberate); TSAT (tris-(succinimido)aminotriacetate); BS(PEG)5 (PEGylated bis(sulfosuccinimidyl)suberate); BS(PEG)9 (PEGylated bis(sulfosuccinimidyl)suberate); DSP (dithiobis(succinimidyl propionate)); DTSSP (3,3′-dithiobis(sulfosuccinimidyl propionate)); DST (dithiosuccinimidyl tartrate); BSOCOES (bis(2-(succinimidyloxycarbonyloxy)ethyl) sulfone); EGS (ethylene glycol bis(succinate succinimidyl ester)); DMA (dimethyl adipimidate); DMP (dimethyl pimelimidate); DMS (dimethyl suberimidate); DTBP (Wang and Richard’s Reagent); BM(PEG)2 (1,8-bismaleimido-diethylene glycol); BM(PEG)3 (1,11-bismaleimido-triethylene glycol); BMB (1,4-bismaleimidobutane); DTME (dithiobismaleimidoethane); BMH (bismaleimidohexane); BMOE (bismaleimidoethane); TMEA (tris(2-maleimidoethyl)amine); SPDP (3-(2-pyridyldithio)propionic acid succinimidyl ester); SMCC (trans-4-(maleimidomethyl)cyclohexane-1-carboxylic acid succinimidyl ester); SIA (succinimidyl iodoacetate); SBAP (3-(bromoacetamido)propionic acid succinimidyl ester); STAB (succinimidyl (4-iodoacetyl)aminobenzoate); sulfo-SIAB (sulfosuccinimidyl (4-iodoacetyl)aminobenzoate);AMAS (N-α-maleimidopropionyloxy succinimide ester); BMPS (N-β-maleimidopropionyloxy succinimide ester); GMBS (N-γ-maleimidobutyryloxy succinimide ester); Sulfo-GMBS (N-γ-maleimidobutyryloxy sulfo succinimide ester); MBS (m-maleimidobenzoyl-N-hydroxysuccinimide ester); Sulfo-MBS (m-maleimidobenzoyl-N-hydroxysulfo succinimide ester); SMCC (4-(N-maleimidomethyl) cyclohexane-1-carboxylic acid succinimide ester); Sulfo-SMCC (4-(N-maleimidomethyl) cyclohexane-1-carboxylic acid sulfo succinimide ester); EMCS (N-ε-maleimidohexanoyloxy succinimide ester); Sulfo-EMCS (N-ε-maleimidohexanoyloxy sulfo succinimide ester); SMPB (4-(p-maleimidophenyl) butyric acid succinimide ester); Sulfo-SMPB (4-(N-maleimidophenyl) butyric acid sulfo succinimide ester); SMPH (6-((β-maleimidopropionylamino) hexanoic acid succinimide ester)); LC-SMCC (Succinimide 4-(N-maleimidomethyl) cyclohexane-1-carboxyl-(6-aminohexanoate)); Sulfo-KMUS (N-κ-maleimidoundecanoyloxy sulfo succinimide ester); SPDP (3-(2-pyridyldithio) propionic acid succinimide ester); LC-SPDP (6-(3(2-pyridyldithio) propionylamino) hexanoic acid succinimide ester); LC-SPDP (6-(3(2-pyridyldithio) propionylamino) hexanoic acid succinimide ester); Sulfo-LC-SPDP (6-(3′-(2-pyridyldithio) propionylamino) hexanoic acid sulfo succinimide ester); SMPT (4-succinimidyloxycarbonyl-α-methyl-α(2-pyridyldithio) toluene); PEG4-SPDP (PEGylated long-chain SPDP crosslinker); PEG12-SPDP (PEGylated long-chain SPDP crosslinker); SM(PEG)2 (PEGylated SMCC crosslinker); SM(PEG)4 (PEGylated SMCC crosslinker); SM(PEG)6 (PEGylated long-chain SMCC crosslinker); SM(PEG)8 (PEGylated long-chain SMCC crosslinker); SM(PEG)12 (PEGylated long-chain SMCC crosslinker); SM(PEG)24 (PEGylated long-chain SMCC crosslinker); BMPH (N-β-maleimidopropionic acid hydrazide); EMCH (N-ε-maleimidohexanoic acid hydrazide); MPBH (4-(4-N-maleimidophenyl) butyric acid hydrazide); KMUH (N-κ-maleimidoundecanoic acid hydrazide); PDPH (3-(2-pyridyldithio) propionic acid hydrazide);ATFB-SE (4-azido-2,3,5,6-tetrafluorobenzoic acid, succinimidyl ester); ANB-NOS (N-5-azido-2-nitrobenzoyloxy succinimide); SDA (NHS-diazirine) (4,4'-azidovaleric acid succinimidyl ester); LC-SDA (NHS-LC-diazirine) (6-(4,4'-azidovaleramido)hexanoic acid succinimidyl ester); SDAD (NHS-SS-diazirine) (2-((4,4'-azidovaleramido)ethyl)-1,3'-dithiopropionic acid succinimidyl ester); sulfo-SDA (sulfo-NHS-diazirine) (4,4'-azidovaleric acid sulfo succinimidyl ester); sulfo-LC-SDA (sulfo-NHS-LC-diazirine) (6-(4,4'-azidovaleramido)hexanoic acid sulfo succinimidyl ester); sulfo-SDAD (sulfo-NHS-SS-diazirine) (2-((4,4'-azidovaleramido)ethyl)-1,3'-dithiopropionic acid sulfo succinimidyl ester); SPB (succinimidyl-[4-(psoralen-8-yloxy)]-butyrate); sulfo-SANPAH (6-(4'-azido-2'-nitrophenylamino)hexanoic acid sulfo succinimidyl ester); DCC (dicyclohexylcarbodiimide); EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride); glutaraldehyde; formaldehyde; and combinations or derivatives thereof.;

[0199] Linkers can also be used to attach molecules to substrates. The linker can have any useful number of functional groups or reactive groups and can be monofunctional (having one functional group), bifunctional, trifunctional, tetrafunctional or contain a greater number of functional groups. In some cases, heterobifunctional linkers can be used to attach molecules (e.g., nucleic acid molecules, peptides or polymers) to substrates. The heterobifunctional linker can contain any useful functional groups as described herein.Non-limiting examples of heterobifunctional linkers include: p-azidobenzoyl hydrazide (ABH), N-5-azido-2-nitrobenzoyloxy succinimide (ANB-NOS), N-[4-(p-azidosalicylamido)butyl]-3'-(2'-pyridyldithio)propanamide (APDP), p-azidophenyl glyoxal monohydrate (APG), bis[B-(4-azidosalicylamido)ethyl] disulfide (BASED), bis[2-(succinimidyloxycarbonyloxy)ethyl] sulfone (BSOCOES), BMPS, 1,4-bis[3'-(2'-pyridyldithio)propanamido]butane (DPDPB), dithio-bis(succinimidyl propionate) (DSP), disuccinimidyl suberate (DSS), dithio-bis(succinimidyl tartrate) (DST), 3,3'-dithio-bis(sulfosuccinimidyl propionate) (DTSSP), EDC, ethylene glycol bis(succinimidyl succinate) (EGS), N-(E-maleimidocaproic acid hydrazide (EMCH), N-(E-maleimidocaproyloxy)-succinimidyl ester (EMCS), N-maleimidobutyryloxy succinimidyl ester (GMBS), hydroxylamine-HCl, MAL-PEG-SCM, m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), N-hydroxysuccinimide-4-azidosalicylic acid (NHS-ASA), PDPH, N-succinimidyl bromoacetate (SBA), SIA, sulfo-SIA, 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid succinimidyl ester (SMCC), 4-(p-maleimidophenyl)butyric acid succinimidyl ester (SMPB), succinimidyl-6-[B-maleimidopropionamido]hexanoate (SMPH), 3-[2-pyridyldithio]-propionic acid N-succinimidyl ester (SPDP), sulfo-LC-SPDP, N-(p-maleimidophenyl isocyanate (PMPI), N-succinimidyl (4-iodoacetyl)aminobenzoate (SIAB), sulfo-MBS, sulfo-SANPAH, sulfo-SMCC, sulfo-DST, sulfo-EMCS, sulfo-GMBS, N-hydroxysulfosuccinimide-4-azidobenzoate (sulfo-HSAB), (4-azidophenyl)-1,3-dithiopropionic acid sulfosuccinimidyl ester (sulfo-SADP), 2-(m-azido-o-nitrobenzamido)-ethyl-1,3'-dithiopropionic acid sulfosuccinimidyl ester (sulfo-SAND), sulfosuccinimidyl-2-(p-azidosalicylamido)ethyl-1,3-dithiopropionate (sulfo-SASD), sulfo-SIAB, sulfo-SMCC, sulfo-SMPB, etc.

[0200] Additional examples of conjugation reactions that can be used to attach molecules to substrates include the Ullmann reaction, Heck reaction, Negishi reaction, Stille reaction, Suzuki reaction, Buchwald-Hartwig coupling, Castro-Stevens coupling, Glaser coupling, Kumada coupling, Larock indole synthesis, Miyaura boration, Sonagashira cross-coupling, Grubbs reaction.

[0201] More than one type of molecule can be coupled to a substrate. For example, a substrate can be coupled to a nucleic acid molecule and a peptide. Alternatively, a substrate can be coupled to only one type of molecule (e.g., only a nucleic acid molecule, only a peptide, only a lipid, only a carbohydrate, etc.). A substrate can be coupled to any useful combination of molecules, linkers, reactive moieties, or functional groups, which can be coupled at any useful density, as described elsewhere herein. For example, a multifunctional linker can be used to attach both a nucleic acid barcode molecule and a peptide to a substrate. Alternatively, a substrate can contain multiple bifunctional linkers that can conjugate to different molecules. In another example, a substrate can contain a linker and a reactive site; the linker can be used to attach one type of molecule (e.g., a peptide or a nucleic acid molecule), while the reactive site can be used to attach another type of molecule (e.g., a nucleic acid molecule or a peptide).

[0202] A linker can contain other functional moieties such as a spacer (e.g., a polymer chain, such as PEG, an alkyl chain, etc.), a cleavage site (e.g., a disulfide bond that can be cleaved upon application of a chemical stimulus, a photocleavable or thermocleavable moiety, etc.), an enzyme recognition site, etc.

[0203] Various methods (e.g., self-assembled monolayers, patterning methods, linking moieties, etc.) can be used to control the proximity of a molecule conjugated to a substrate to its nearest neighboring molecule (e.g., another molecule). In some cases, it can be advantageous for two molecules (e.g., two polymerizable molecules, such as a peptide and a nucleic acid molecule, or two nucleic acid molecules) to have a close proximity. For example, with respect to the sequencing methods described herein, a capture moiety can be used to conjugate monomers of a polymeric analyte, and after monomer cleavage, additional polymerizable molecule(s) or multiple polymerizable molecules in proximity to the capture moiety may be required to allow transfer of information encoded by the polymerizable molecules of the binder. The proximity of molecules (e.g., a capture moiety and a polymerizable molecule) can be mediated using tethering molecules such as nucleic acid "staples" or multifunctional linkers.

[0204] Nucleic acid molecules can be coupled to a substrate by direct coupling. In such cases, the substrate or the nucleic acid molecule can contain functional moieties that can interact with each other. For example, the substrate and the nucleic acid molecule can contain complementary click chemistry pairs, such as alkynes and azides. In one such example, the substrate can contain an alkyne moiety (e.g., DBCO), which can react with an azide-functionalized nucleic acid molecule. The nucleic acid molecule can react with the alkyne moiety in a click chemical reaction to covalently link the substrate to the nucleic acid molecule. In another example, the substrate can contain an avidin or streptavidin moiety, with which a biotinylated nucleic acid molecule can interact and bind non-covalently. Alternatively or additionally, the substrate can contain a nucleic acid molecule to which an additional nucleic acid molecule (e.g., a nucleic acid analyte, a nucleic acid linker) is conjugated using hybridization, ligation, click chemistry, crosslinking (e.g., photocrosslinking such as CNVK).

[0205] Alternatively or additionally, a nucleic acid molecule can be coupled to a substrate using a linker, for example, as described elsewhere herein. The linker can contain at least two functional groups (e.g., a heterobifunctional linker) that can couple to both the substrate and the nucleic acid molecule. In an example, the substrate can contain an amine group, and an alkyne-functionalized DNA primer (e.g., a DBCO-DNA primer) can be attached using a linker such as N-hydroxysuccinimide ester of azidoacetic acid. In another example, an amine-functionalized substrate can be coupled to an azide-functionalized DNA primer using a DBCO-NHS ester or a DBCO-PEG-NHS ester linker. As described elsewhere herein, the linker can contain additional functional moieties (e.g., a cleavage site, a spacer such as a polymer or an alkyl chain).

[0206] Similarly, a peptide can be coupled to a substrate by direct coupling or by using a linker. The peptide can be coupled to the substrate at the end of the peptide (e.g., the C-terminus or the N-terminus), at an internal residue or amino acid of the peptide, or at multiple positions along the peptide. In an example of direct coupling, the peptide can be functionalized with a moiety that can interact with a moiety of the substrate (e.g., a click chemistry pair, avidin-biotin). For example, the substrate and the peptide can contain complementary click chemistry pairs, such as alkynes and azides, or binding partners such as avidin and biotin. In one example of a click chemistry pair, the substrate can contain an alkyne moiety (e.g., DBCO), which can react with an azide-functionalized peptide. The peptide can react with the alkyne moiety in a click chemical reaction to covalently link the substrate to the peptide. In another example, the substrate can contain an avidin or streptavidin moiety, with which a biotinylated peptide can interact and bind non-covalently.

[0207] Alternatively or additionally, a linker can be used to couple the peptide to the substrate, e.g., as described elsewhere herein. The linker can comprise at least two functional groups (e.g., a heterobifunctional linker) that can couple to both the substrate and the nucleic acid molecule. In an example, the substrate can comprise an amine group, and the alkyne-functionalized peptide can be attached using a linker such as NHS ester of azidoacetic acid. In another example, an amine-functionalized substrate can be coupled to an azide-functionalized peptide using a DBCO-NHS ester or a DBCO-PEG-NHS ester linker. In yet another example, an EDC and sulfo-NHS can be used to couple a substrate comprising an amine group to an azide-functionalized peptide.

[0208] Peptides can be functionalized with functional moieties to enable attachment or coupling of the peptides to substrates. The functional moieties can include silanes such as aminosilanes (e.g., APTES), amino-PEG-silanes, click chemistry moieties, or other linking moieties and can be attached to the peptide at the peptide terminus (N-terminus or C-terminus), at internal amino acids, or at multiple positions (e.g., multiple internal amino acids, one or both termini, etc.). Chemical methods for functionalizing peptides can include using photoredox-catalyzed C-terminus-specific conjugation (e.g., via C-terminal decarboxylative alkylation), such as described by Bloom et al., Nature Chemistry 10, 205–211, 2018, and Zhang et al., ACS Chem. Biol. 2021, 16, 11, 2595–2603, each of which is incorporated herein by reference in its entirety, or amide coupling with an amine-functionalized surface. N-terminus attachment can include coupling the N-terminal amine group to a carboxyl-functionalized surface or using 2-pyridinecarboxaldehyde variants. Alternatively or additionally, functionalization of the termini of the peptide can be achieved enzymatically or using enzyme analogs such as ribozymes or deoxyribozymes. In examples of enzymatic functionalization and attachment, carboxypeptidase or amidase is used for C-terminus functionalization (e.g., as described in Xu et al., ACS Chem. Biol. 2011 Oct 21; 6(10):1015–1020; Zhu et al., Chinese Chemical Letters. 2018, Vol. 29, No. 7, pp. 1116–1118; and Zhu et al., ACS Catal. 2022, 12, 13, 8019–8026, each of which is incorporated herein by reference in its entirety), which can allow addition of a click chemistry moiety to the peptide. Then, the click chemistry-functionalized peptide can be directly attached to the substrate via another clickable group (e.g., BCN-azide or DBCO-azide coupling), or in other cases, can react with another linker or polymerizable molecule (e.g., a decoy nucleic acid molecule bearing a clickable group), and then the linker or polymerizable molecule can be directly or indirectly linked to the substrate (e.g., using a capture nucleic acid molecule and hybridizing with the decoy nucleic acid molecule). Additional examples of enzymes that can be used for functionalization or attachment include sortase A, subtiligase, Butelase I, or trypsiligase. In some examples, ubiquitin ligase can be used to attach a ubiquitin protein bearing a linker moiety to a substrate. Then, these linker moieties can be used for chemical attachment of the substrate to which the protein is conjugated with ubiquitin. In some examples, glycosylases can be used to conjugate functionalized sugar groups (e.g., click chemistry-functionalized sugars, polymer-conjugated sugars, biotinylated sugars) to amino acid residues, which can allow attachment to substrates (e.g., via click chemistry, polymer crosslinking, or nucleic acid hybridization, avidin-biotin interaction), etc.Internal amino acid residues or post-translationally modified residues can be coupled to the substrate using, for example, thiol labeling, amide coupling of glutamic or aspartic acid residues using EDC / NHS chemistry or DMT-MM, esterification of glutamic or aspartic acid residues, alkylation of cysteine or disulfide labeling, or amide coupling to lysine residues.

[0209] The peptide can be treated before, during, or after coupling to the substrate. In some cases, the peptide is conjugated to a tag that enables attachment to the substrate, for example, using a His tag, SNAP tag, CLIP tag, capturer, spy tag, nucleic acid tag (e.g., a bait oligonucleotide for a capture oligonucleotide that can be attached to the substrate).

[0210] In some examples, it may be advantageous to block or protect primary amine or carboxyl groups and optionally de-block or de-protect the N-terminal primary amine or C-terminal carboxyl group to facilitate N-terminal or C-terminal attachment to the substrate. In an example, site-specific (e.g., C-terminal) attachment of the peptide can be achieved by reacting the peptide with a linker containing an amine-reactive group (e.g., isothiocyanate such as PITC) and a reactive group (e.g., a click chemistry group). The linker can be, for example, a PITC-conjugated click chemistry moiety such as PITC-azide, PITC-alkyne, optionally with a spacer moiety in between, e.g., PITC-alkyl-azide, PITC-PEG-azide, PITC-alkyl-alkyne, PITC-PEG-azide). The linker reacts with the primary amine and “blocks” the primary amine (e.g., modifies lysine), including the N-terminal. Subsequent cleavage of the N-terminal amino acid can be performed (e.g., using an Edman reagent such as an acid), and one of the remaining modified lysines can be attached to the substrate (e.g., using a click chemistry moiety conjugated to an amine-reactive group). Optionally, the peptide can be treated with a protease, for example, LysC, which cleaves the peptide such that the remaining peptide has a C-terminal lysine and such that the remaining peptide contains a primary amine only at the C-terminal lysine residue and the N-terminal; such cleavage can be performed before reacting the amine-reactive group, for example, as shown by Xie et al. Langmuir 2022, 38, 30, 9119–9128, which is incorporated herein by reference in its entirety.

[0211] Similarly, carboxyl groups can be reacted in a manner that enables C-terminal or internal residue attachment. In an example of C-terminal conjugation, when treated with an activating reagent (e.g., acetic anhydride) to generate a peptide-thiohydantoin (at the C-terminus) and "blocked" carboxyl groups on aspartic and glutamic acid residues, the carboxyl groups can be labeled with a C-terminal sequencing reagent such as isothiocyanate. The thiohydantoin can then be reacted to couple with a substrate. Alternatively, via a single round of C-terminal sequencing degradation or via protease cleavage of the C-terminal amino acid, only a single reactive carboxyl is exposed at the C-terminal amino acid. The single reactive C-terminal carboxyl can then be used as the reactive moiety of a single attachment site.

[0212] In another method, peptides or proteins can be attached via the N-terminus using the specific reactivity of the N-terminal amine group. Amine-based reactions, such as amide coupling, can be carried out at low pH where only the N-terminal amine group is active. Additionally, 2-pyridinecarboxaldehyde and variants can be used to react with the N-terminal amine group.

[0213] In some cases, polymerization reactions can be used to conjugate peptides to substrates, e.g., radical polymerization such as Michael-type addition using PEGylated peptides, methacrylamide-modified peptides, maleimide-capped oligo NIPAAM-conjugated peptides; photocrosslinking of phenylazide-conjugated peptides or other polymerization reactions with monomer-conjugated peptides, e.g., as described by Krishna et al. Biopolymers. 2010;94(1):32-48, which is incorporated herein by reference in its entirety.

[0214] A variety of types of molecules can be attached to a substrate. The substrate can include any combination of molecules conjugated thereto, including but not limited to peptides, proteins (e.g., enzymes, ribozymes, deoxyribozymes, antibodies, nanobodies, antibody fragments), nucleic acid molecules, lipids, carbohydrates or sugars, metabolites, small molecules, polymers, metals, virus particles, biotin, avidin, streptavidin, neutravidin, etc. A variety of types of molecules can be attached to the substrate simultaneously or in a sequential manner. For example, the substrate can be treated to conjugate a nucleic acid molecule and then treated to conjugate a peptide, or alternatively, the substrate can be treated to conjugate a peptide before the nucleic acid molecule. Any number of conjugations or attachments can be used. For example, in cases where multiple types of molecules are attached to the substrate, any number of conjugation chemistries can be used for each type of molecule.

[0215] The substrate or a portion thereof can be subjected to conditions sufficient to passivate the substrate or a portion thereof. Passivation of the substrate can be used for various purposes, such as preventing non-specific binding of binders, altering the surface density of molecules (e.g., increasing the density of nucleic acid molecules or peptides), blocking reaction sites (e.g., blocking available click chemistry moieties after molecules are conjugated to the substrate), etc. Passivation can be achieved using chemical methods, e.g., depositing blockers such as proteins (e.g., albumin), Tween-20, polymers, metals or metal oxides, or using biochemical methods, e.g., using metal microorganisms. Substrates containing reactive moieties can also be passivated after molecular conjugation (e.g., coupling of nucleic acid molecules, peptides, etc.) by reacting any unreacted sites with appropriate molecules. For example, a substrate containing a click chemistry moiety (e.g., DBCO beads) can be coupled to a molecule of interest (e.g., a polymerizable molecule such as a nucleic acid molecule, a peptide) at a useful density using click chemistry (e.g., azide-nucleic acid molecule, azide-peptide). Unreacted sites can be passivated by providing complementary click chemistry molecules and reacting the complementary click chemistry molecules (e.g., azide-polymer (e.g., PEG-azide)), which can reduce downstream non-specific interactions.

[0216] Substrate passivation can occur at any useful time or step. For example, passivation for blocking unreacted DBCO sites can be performed before, during, or after conjugation of an analyte or other molecule of interest (e.g., peptides and nucleic acid molecules). Passivation can be controlled by the stoichiometry or density of the passivating agent relative to the molecule of interest, or by physical methods (e.g., photopatterning, self-assembled monolayers, etc.).

[0217] Sample Processing

[0218] The present disclosure also provides methods for processing samples. One or more methods for processing samples can include preparing a biological sample for analysis, which in some cases includes cell partitioning for performing single cell analysis. Methods for processing biological samples can include extracting or isolating one or more peptides or proteins from the biological sample for further processing and analysis, as described elsewhere herein.

[0219] Preparation of cell suspensions for single cell analysis: The methods described herein can involve preparing a single cell suspension from a biological sample. A single cell suspension can be prepared from a biological sample by dissociating the cells and optionally culturing them in a liquid medium. In some cases, the biological sample includes a liquid sample. For example, the biological sample can include a bacterial liquid culture, a mammalian liquid culture, blood, plasma, or serum sample. Processing of such liquid samples can include centrifugation (e.g., to separate the cells), resuspending the cells in a suitable medium (e.g., Dulbecco's phosphate buffered saline), and optionally culturing the isolated cells.

[0220] Biological samples can include cultured cells, such as cells in suspension culture, or cells adhered to a solid surface, such as a Petri dish or tissue culture dish. Cultured adherent cell samples can be processed to produce a cell suspension, for example, via a protease (such as trypsin), to detach the cells from the surface. Biological samples can include tissue or biopsy samples. Tissue or biopsy samples can be processed mechanically or enzymatically to produce a cell suspension. Such processing can include sonication (mechanical processing) or enzymatic treatment, such as using pronase, collagenase, hyaluronidase, metalloproteinase, trypsin, or other enzymes that digest extracellular matrix components. The dissociated cells can then be stored in a suitable buffer, such as DPBS.

[0221] Cell sorting: Biological samples or cell suspensions can be sorted to isolate cells of interest. Sorting can be based on the mass or characteristics of the cells (e.g., expression of a protein target, size, deformability, fluorescence, or other optical properties, or other physical properties of the cells) to select or isolate cells. Sorting can be accomplished using any number of methods, such as using immunoselection (e.g., fluorescence-activated cell sorting (FACS) or magnetic-activated cell sorting (MACS)), electrophoretic methods, chromatography, microfluidic methods (e.g., using inertial focusing, cell traps, electrophoresis, isoelectric focusing), acoustic sorting, optical sorting (e.g., optical tweezers), mechanical cell picking (e.g., using a manual or robotic pipette), or passive methods (e.g., gravity sedimentation).

[0222] Partitioning: The cells of a biological sample or cell suspension can be separated into individual partitions such that at least a subset of the individual partitions contains single cells. The individual partitions can contain barcode molecules (e.g., fluorophores or fluorophore sets, nucleic acid barcode molecules, etc.). The barcode molecules can be unique to the partition such that each individual partition contains a barcode sequence that is different from other partitions. The barcode molecules can be loaded into the individual partitions at any useful ratio of barcode molecule to sample type (e.g., cells, proteins, nucleic acid molecules). The barcode molecules can be loaded into the partitions such that about 0.0001, 0.001, 0.1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 500, 1000, 5000, 10000, or 200000 barcodes are loaded per sample type. In some cases, the barcodes are loaded into the partitions such that more than about 0.0001, 0.001, 0.1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 500, 1000, 5000, 10000, or 200000 barcodes are loaded per sample type. In some cases, the barcodes are loaded into the partitions such that less than about 0.0001, 0.001, 0.1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 500, 1000, 5000, 10000, or 200000 barcodes are loaded per sample type.

[0223] The partitions can assume any useful geometry, such as droplets, micro-wells, solid substrates, gels (e.g., cells encapsulated in gel beads), beads, flasks, tubes, spots, capsules, channels, chambers, or other compartments or containers. The partitions can be part of an array of partitions, such as droplets in a microfluidic device, micro-wells in a microplate, spots on a multi-spot array, etc.

[0224] Lysis, permeabilization, and analyte extraction: Single cells (e.g., in partitions) can be processed to obtain one or more analytes contained therein. Methods for processing single cells can include lysing the cells to release the contents into separate compartments or partitions. Lysis can be performed using detergents (e.g., Triton-X 100, sodium dodecyl sulfate, sodium deoxycholate, CHAPS), RIPA buffer, temperature changes (e.g., elevated or lower temperature, freezing, freeze-thaw), enzymes, ribozymes, deoxyribozymes, mechanical lysis (e.g., sonication, application of mechanical force), electro-lysis, or combinations thereof. Lysis can be performed in the presence of protease inhibitors to prevent degradation or digestion of proteins from the cells. The contents can optionally be further processed, such as subjected to purification or extraction, denaturation of proteins or peptides, enzymatic or chemical digestion, etc. In some cases, the contents can be enzymatically digested to remove nucleic acid molecules, e.g., using nucleases such as DNase or RNase. Alternatively or additionally, the cells can be fixed (e.g., using fixatives) and / or permeabilized. Examples of fixatives include aldehydes (e.g., glutaraldehyde, formaldehyde, paraformaldehyde), alcohols (e.g., methanol, ethanol), acetone, acids (e.g., acetic acid, Davidson’s AFA), oxidants (e.g., osmium tetroxide, potassium dichromate, chromic acid, permanganate), Zenker fixative, picrate, Hepes-glutamate buffer-mediated organic solvent protection effect (HOPE), or Karnovsky fixative. Cell permeabilization can be achieved mechanically (e.g., using sonication, electroporation, shearing) or chemically (e.g., using organic solvents such as methanol or acetone, or detergents such as saponin, Tween-20, Triton X-100).

[0225] Protein processing: A biological sample (or single cell suspension or partitioned cell) can be further processed to enable proteomic analysis. For example, protein depolymerization in the sample can be carried out, such as using chemical or mechanical methods. Chemical depolymerization methods can include but are not limited to sodium dodecyl sulfate (SDS), Triton-X100, 3-((3-cholamidopropyl)dimethylammonio)-1-propanesulfonate (CHAPS), ethylene carbonate, or formamide. Mechanical depolymerization methods can include but are not limited to sonication or heat treatment. The biological sample (or single cell suspension or partitioned cell) can be subjected to conditions sufficient to denature one or more proteins. Denaturation can be achieved using heat, chemicals (such as SDS, urea, guanidine, formamide, metal-organic compounds), reducing agents (such as dithiothreitol (DTT), β-mercaptoethanol, TCEP), urea, chaotropes, enzymes (such as ClpX, ClpS, unfoldase), ribozymes or deoxyribozymes. Similarly, a peptide or protein can be subjected to conditions that dissolve the peptide or protein in solution, such as using detergents, organic solvents, spermidine, or tagging the peptide or protein with a multi-ion tag (such as DNA, PEG, or other polymers). Alternatively or additionally, the peptide or protein can be enriched or purified; in an example, the peptide or protein of interest can be precipitated using trichloroacetic acid, chloroform, TRIzol, or other chemical reagents. Other biological or chemical reagents can be included during protein processing, such as lysozyme, papain, cruzain, trypsin, protease inhibitors, nucleases, or proteins containing nucleases (such as DNase, RNase, DNA glycosylase, restriction endonuclease, transposase, micrococcal nuclease, Cas protein). In some cases, minimal protein processing is carried out, for example, to maintain the native state or conformation of the protein.

[0226] Peptides or proteins can be fragmented prior to analysis. Fragmenting the protein can be used to reduce the size of the protein and allow for efficient processing of the peptides, as described elsewhere herein. Proteases (such as trypsin, chymotrypsin, pepsin, Lys-C, Glu-C, proteinase K, furin, thrombin, endopeptidase, papain, subtilisin, elastase, enterokinase, genenanse, endoproteinase, metalloproteinase), or chemical treatment (such as cyanogen bromide, hydrazine, hydroxylamine, formic acid, BNPS-skatole, iodosylbenzoic acid, 2-nitro-5-thiocyanatobenzoic acid, etc.) can be used for fragmentation. Alternatively or additionally, fragmentation can be carried out using mechanical methods, such as sonication, vortexing, mechanical stirring, using temperature changes (such as freeze / thaw, heating), or other fragmentation methods.

[0227] Enrichment of proteins or peptides in biological samples can be carried out, for example, for separating proteins and peptides from cell debris or other types of analytes (e.g., nucleic acids, lipids, carbohydrates, metabolites). Such enrichment can include, for example, using affinity columns (e.g., ion exchange), size exclusion columns, affinity precipitation (e.g., immunoprecipitation), chemical precipitation (e.g., using trichloroacetic acid, chloroform, TRIzol), chromatography (e.g., HPLC), or electrophoresis. In cases where cells are partitioned prior to enrichment, beads, affinity microcolumns, affinity beads, etc. can be used for enrichment. In some cases, proteins or peptides can be fractionated, which can be used to separate proteins by size, hydrophobicity, charge, affinity, size, mass, density, etc.

[0228] Peptides can be barcoded in bulk or in partitions. Peptides can be barcoded with any useful type of barcode molecule (e.g., spectral or fluorescent barcodes, mass tags, nucleic acid barcode molecules, etc.). Barcode molecules can allow for the identification of the originating peptide, partition, sample, cell, or cell compartment. For example, a cell sample can be partitioned such that the partition contains at most one cell; the partition can contain a unique barcode molecule (e.g., a nucleic acid barcode molecule) that identifies the partition and thus the cell. Subsequently, labeling the peptides within the partition with a barcode molecule (e.g., by permeabilizing or lysing the cell) can be used to identify the peptides as having originated from or being from the same cell or partition. In other examples, the substrate can contain a nucleic acid molecule that contains a unique barcode sequence different from the barcode sequences of other substrates. Thus, the barcode sequence can be used to identify the substrate. In some cases, a cell sample can be used to partition the barcoded substrate such that at least a subset of the partitions includes single cells and single barcoded substrates. Thus, peptides generated from a single cell and transferred to the barcoded substrate can all be identified as originating from that single cell. Barcode molecules can contain additional useful functional sequences, such as UMI, primer sites, restriction sites, cleavage sites, transposon sites, sequencing sites, read sites, etc.

[0229] Any suitable chemistry can be used to achieve the attachment of the barcode molecule to the peptide. For example, C-terminal conjugation of nucleic acid barcode molecules can be achieved by coupling an amine-conjugated DNA barcode molecule amide to the peptide or by thiol alkylation (e.g., reacting a thiolated peptide with an alkylated (e.g., iodoacetamide) DNA barcode molecule). N-terminal conjugation can be achieved, for example, using 2-pyridinecarboxaldehyde labeling of the DNA barcode and reacting with the N-terminus of the peptide. Internal residues, such as glutamate, can also be labeled with an amine-conjugated DNA barcode molecule or a carboxylated DNA barcode (e.g., to react with the primary amine in lysine).

[0230] For a given peptide, individual peptides can be barcoded at multiple positions. The peptides can be labeled at multiple sites with the same or different barcode sequences. For example, peptides can be partitioned into partitions containing multiple identical barcode molecules, the barcode molecules containing barcode sequences unique to that partition. The peptides can be labeled at single or multiple sites with unique partition barcode sequences, optionally each including a unique molecular identifier (UMI), such that subsequent downstream analysis (e.g., sequencing) can be attributed to the same peptide using the barcode sequence. In some cases, the termini (e.g., N-terminus or C-terminus) or internal amino acids of the peptide can be labeled with barcodes. In some cases, the peptides can be fragmented prior to analysis or sequencing; thus, multiple identical barcode molecules attached upstream to the same peptide can allow sequence analysis to be attributed back to a single peptide. Barcoding of the peptides can occur before, during, or after fragmentation. The peptides can be labeled with barcodes (e.g., nucleic acid barcode molecules) using any suitable chemistry (e.g., as described above) or using bifunctional or trifunctional linkers containing multiple linking moieties (e.g., as described elsewhere herein, such as click chemistry moieties, NHS-esters, EDC, etc.). For example, C-terminal attachment can include amide coupling to the C-terminal carboxyl group or photoredox tagging of the C-terminal carboxyl group (e.g., addition of an electrophilic tag). N-terminal attachment can include amide coupling to the N-terminal amino group, where specific attachment can occur at low pH, or use of a 2-pyridinecarboxaldehyde variant for specific attachment to the N-terminus. Internal attachment can include, for example, amide coupling using EDC / NHS chemistry or DMT-MM to glutamic or aspartic acid; alkylation or disulfide tagging of cysteine; or amide coupling to lysine residues.

[0231] In some examples, peptides can be labeled with different barcode molecules, which can be indexed by their proximity to each other. For example, primers that can anneal to neighboring barcode molecules can be used. In one such method, after a protein has been labeled with multiple barcodes having different barcode sequences, proximity-based polymerase extension can be used to copy and associate the sequences of neighboring barcodes. For example, each barcode molecule can contain a primer binding site, and a dual-primer linker sequence containing two sequences can anneal to the primer binding site. The dual-primer linker sequence can bind to the primer binding sites of two neighboring barcodes. An extension reaction, such as using a polymerase, can extend and copy the barcode sequences of the neighboring barcodes. Subsequently, the dual-primer linker sequences that now have copies of two neighboring barcodes can be removed and sequenced. From the sequencing reads, an adjacency matrix of the barcode sequences can be generated (e.g., corresponding to the barcode sequences on a single dual-primer linker that are spatially proximal). Thus, each barcode sequence can be associated with nearby neighboring barcode sequences, and therefore, peptide portions can be aligned or grouped as neighboring. Such methods can be useful in cases where a peptide is fragmented, such that individual fragments of the peptide can be made to correspond to nearest neighbor fragments using the barcode sequences.

[0232] In another example, bridge amplification can be used to barcode a peptide at multiple positions of a given peptide. In such methods, a peptide or protein can be labeled with nucleic acid primers at multiple sites. Nucleic acid barcode molecules can be provided that can anneal to (not shown) or be ligated to the nucleic acid primers. Subsequent rounds of bridge amplification can be performed to copy the nucleic acid barcode molecules to other primers located at other sites of the given peptide. In some examples, a peptide can be tagged with multiple copies of a nucleic acid primer, and the barcode sequences can be provided sparsely such that only one nucleic acid primer per peptide is extended by polymerase extension. Subsequent rounds of bridge amplification can result in peptides having the same barcode sequence at each nucleic acid primer. Subsequent fragmentation of the peptide can be performed such that peptide fragments on average contain a single barcode. Thus, in some cases, the output of such amplification methods can be peptides with individual barcodes that are generated from fragmenting a multi-labeled protein, where peptides from the same protein have the same barcode.

[0233] A cell sample can be partitioned into separate partitions or compartments (e.g., droplets, microwells) such that at least a subset of the partitions contains single cells. The partitions can then be treated with a lysis agent to lyse the cells and release the proteins from the cells into the partitions. The proteins can then be labeled with partition-specific barcodes (e.g., using barcode beads) such that all peptides or proteins generated from a single compartment contain the same barcode. In some examples, the barcode comprises a nucleic acid barcode molecule, and the barcode sequence can be used in downstream processing (e.g., via sequencing) to identify the partition or cell from which the peptide originated. The nucleic acid barcode molecule can contain any additional useful sequences, such as UMI, primer sequences, etc.

[0234] Bulk processing: Biological samples can be processed in bulk. For example, a biological sample can be processed to obtain a suspension of cells, which can be directly lysed in the suspension without partitioning the cells into separate compartments. Any useful method (e.g., as described above) can be used to lysate the cells in bulk and optionally further processed, such as homogenization, protease inhibition, denaturation, protein processing (e.g., chemical treatment, fragmentation), or combinations thereof. The biological sample can be pre-treated prior to cell lysis or protein extraction. Such pre-treatment can include debris removal, purification, filtration, concentration, or sorting.

[0235] Spatial barcoding: Biological samples can include tissue samples containing multiple cells. Methods that retain spatial information can be used to process the tissue samples (e.g., to identify peptides from individual cells), e.g., using spatial barcodes. For example, a 2D or 3D tissue sample can be provided, and individual cells or locations within the tissue sample can be contacted with a plurality of spatial barcodes (e.g., nucleic acid barcode molecules) containing different barcode sequences. Different barcode sequences can be attributed to specific locations in the 2D or 3D tissue sample, which can correspond to the location of the cells. For example, deterministic methods (such as two-photon patterning) or random methods (such as PCR) can be used to provide spatial barcodes to assign unique spatial barcodes to different segments of the 2D or 3D tissue sample. Thus, peptides labeled with spatial barcodes can be traced back to a single location within the tissue sample, or to a single cell.

[0236] In another example of the spatial barcoding workflow for tissue samples, a tissue sample comprising multiple cells (shown as a 2x2 cell array) can be provided. The tissue sample can be subjected to lysis or fixation and permeabilization to provide access to the proteins contained within the multiple cells. Spatial barcodes, such as nucleic acid barcode molecules, can be provided. The spatial barcodes can include coordinate or location information. In an example, each cell can be contacted with a different spatial barcode, or portions of the cell can be contacted with different spatial barcodes, which can optionally be pre-indexed (e.g., using imaging or deterministic spatial barcoding methods). Further processing of the peptides can be performed as described elsewhere herein. When the peptides are labeled with spatial barcodes, each peptide with a spatial barcode can be returned to its origin coordinate or location, which can help identify the origin cell that produced the peptide.

[0237] In one example, the spatial barcode array can be provided on a substrate (e.g., a glass microscope slide, a hydrogel mesh). In some cases, the spatial barcodes can be directly conjugated to the substrate, or they can be provided on barcoded beads. In an example, multiple beads each containing a different barcode sequence can be arranged in an array on the substrate. Each bead can contain a spatial barcode that includes a spatial barcode sequence and optionally a unique molecular identifier (UMI). The tissue sample (e.g., a fixed tissue sample) can be placed adjacent to (e.g., overlying) the spatial barcode array. The tissue sample can then be subjected to conditions sufficient to transfer the peptides or proteins to the spatial barcode array. For example, the peptides or proteins can be transferred via passive transport (e.g., diffusion or Brownian motion) or via active transport (e.g., electrophoresis, pressure-driven flow, etc.). The peptides or proteins can be attached to the spatial barcodes, for example, using linkers (e.g., containing amine-reactive groups, or click chemistry groups such as azides, alkynes, or other functional moieties such as aldehyde groups or NHS or carboxyl groups), conjugation chemistries, or anchoring agents. Examples of anchoring agents include fixatives such as formaldehyde, paraformaldehyde, glutaraldehyde, or monomers for incorporation into hydrogels such as acryloyl-X, acrylamide, N-(3-aminopropyl)methacrylamide, or N-(3-aminoethyl)methacrylamide, or benzophenone. The anchoring agent can also include multifunctional linkers such as acryloyl-X, biotin-NHS, biotin-PEG-amine, DBCO-NHS, DBCO-amine. For the bead array, multiple beads can be collected from the sample for further processing.

[0238] Computer system

[0239] The present disclosure provides a computer system programmed to implement the methods of the present disclosure. Figure 5Shows computer system 501, which is programmed or otherwise configured to receive sequencing data and output information about the identity of amino acid residues of polymerizable molecules or modified amino acids. Computer system 501 can regulate various aspects of generating the sequencing reads of the present disclosure, for example, receiving one or more sets of sequencing data, processing the sequencing data using algorithms, and outputting one or more sequencing results. Computer system 501 can be the user's electronic device or a computer system remotely located relative to the electronic device. The electronic device can be a mobile electronic device.

[0240] Computer system 501 includes a central processing unit (CPU, also referred to herein as "processor" and "computer processor") 505, which can be a single-core or multi-core processor, or multiple processors for parallel processing. Computer system 501 also includes a memory or storage location 501 (e.g., random access memory, read-only memory, flash memory), an electronic storage unit 515 (e.g., hard disk), a communication interface 520 for communicating with one or more other systems (e.g., network adapter), and peripheral devices 525 (such as caches, other memories, data storage, and / or electronic display adapters). Memory 510, storage unit 515, interface 520, and peripheral devices 525 communicate with CPU 505 via a communication bus (solid line), such as a motherboard. Storage unit 515 can be a data storage unit (or data repository) for storing data. With the aid of communication interface 520, computer system 501 can be operably coupled to a computer network ("network") 530. Network 530 can be the Internet, an intranet, and / or an extranet, or an intranet and / or extranet that communicates with the Internet. In some cases, network 530 is a telecommunications and / or data network. Network 530 can include one or more computer servers that can enable distributed computing, such as cloud computing. In some cases, with the aid of computer system 501, network 530 can implement a peer-to-peer network, which can enable devices coupled to computer system 501 to act as clients or servers.

[0241] CPU 505 can execute a series of machine-readable instructions, which can be embodied in a program or software. The instructions can be stored in a storage location, such as memory 510. The instructions can be directed to CPU 505, which can then program or otherwise configure CPU 505 to implement the methods of the present disclosure. Examples of operations performed by CPU 505 can include fetching, decoding, executing, and writing back.

[0242] CPU 505 can be part of a circuit (such as an integrated circuit). One or more other components of system 501 can be included in the circuit. In some cases, the circuit is an application-specific integrated circuit (ASIC).

[0243] The storage unit 515 can store files, such as drivers, libraries, and saved programs. The storage unit 515 can store user data, such as user preferences and user programs. In some cases, the computer system 501 can include one or more additional data storage units external to the computer system 501, such as on a remote server located to communicate with the computer system 501 via an intranet or the Internet.

[0244] The computer system 501 can communicate with one or more remote computer systems via the network 530. For example, the computer system 501 can communicate with a user's remote computer system. Examples of remote computer systems include personal computers (e.g., portable PCs), tablets or tablet PCs (e.g., iPad, Galaxy Tab), telephones, smart phones (e.g., iPhone, Android - enabled devices, ) or personal digital assistants. A user can access the computer system 501 via the network 530.

[0245] The methods described herein can be implemented by machine - executable code (e.g., on the memory 510 or the electronic storage unit 515) stored at an electronic storage location of the computer system 501 and executable by a machine (e.g., a computer processor). The machine - executable or machine - readable code can be provided in the form of software. During use, the code can be executed by the processor 505. In some cases, the code can be retrieved from the storage unit 515 and stored on the memory 510 for ready access by the processor 505. In some cases, the electronic storage unit 515 can be excluded and the machine - executable instructions are stored on the memory 510.

[0246] The code can be pre - compiled and configured for use with a machine having a processor suitable for executing the code or can be compiled during runtime. The code can be provided in a programming language selected such that the code can be executed in a pre - compiled or just - in - time compiled manner.

[0247] Aspects of the systems and methods provided herein, such as computer system 501, may be embodied in programming. Various aspects of the technology may be considered a "product" or "article of manufacture", typically carried or embodied in a type of machine-readable medium in the form of machine (or processor) executable code and / or associated data. The machine executable code may be stored on an electronic storage unit, such as a memory (e.g., read-only memory, random access memory, flash memory) or a hard disk. A "storage" type medium may include any or all tangible memories of a computer, processor, etc., or associated modules thereof, such as various semiconductor memories, tape drives, disk drives, etc., which may provide non-transitory storage for software programming at any time. All or part of the software can sometimes be communicated via the Internet or various other telecommunications networks. For example, such communication may enable software to be loaded from one computer or processor to another, e.g., from a management server or host to a computer platform of an application server. Thus, another type of medium that can carry software elements includes light, electrical, and electromagnetic waves, such as those used over wired and optical landline networks and over various air links at a physical interface between local devices. Physical elements that carry such waves, such as wired or wireless links, optical links, etc., may also be considered media that carry software. As used herein, unless restricted to non-transitory, tangible "storage" media, the term such as computer or machine "readable medium" refers to any medium that participates in providing instructions to a processor for execution.

[0248] Thus, a machine-readable medium, such as computer-executable code, can take many forms, including but not limited to tangible storage media, carrier media, or physical transmission media. Non-volatile storage media includes, for example, optical or magnetic disks (such as any storage device in any computer, etc.), which can be used to implement databases, etc., as shown in the accompanying drawings. Volatile storage media includes dynamic memory, such as the main memory of such a computer platform. Tangible transmission media includes coaxial cables; copper wire and optical fiber, including the wires that make up the internal bus of a computer system. Carrier transmission media can take the form of electrical or electromagnetic signals, or acoustic or optical waves, such as the acoustic or optical waves generated during radio frequency (RF) and infrared (IR) data communication. Thus, common forms of computer-readable media include, for example: floppy disks, flexible disks, hard disks, magnetic tapes, any other magnetic media, CD-ROMs, DVDs or DVD-ROMs, any other optical media, punched cards, paper tapes, any other physical storage media with a pattern of holes, RAM, ROM, PROM, and EPROM, FLASH-EPROM, any other storage chip or cartridge, carriers that transmit data or instructions, cables or links that transmit such carriers, or any other medium from which a computer can read programming code and / or data. Many of these forms of computer-readable media can participate in transferring one or more sequences of one or more instructions to a processor for execution.

[0249] Computer system 501 can include or communicate with an electronic display 535 that includes a user interface (UI) 540 for providing, for example, sequencing data results, the identity of modified amino acids, the identity of polymerizable molecules, or the nucleic acid sequence of a nucleic acid molecule. Examples of UIs include but are not limited to graphical user interfaces (GUIs) and web-based user interfaces.

[0250] The methods and systems of the present disclosure can be implemented by one or more algorithms. The algorithms can be implemented in software when executed by a central processing unit 505. For example, the algorithms can process sequencing data (e.g., nanopore or nanogap ion current signals) to generate one or more sequencing outputs (e.g., identifying monomers of amino acids or polymerizable molecules, e.g., the nucleic acid sequence of a nucleic acid molecule).

[0251] Examples

[0252] Example 1 - Preparation of a linker for coupling a polymerizable molecule to an amino acid

[0253] As described herein, the linker can comprise two functional components. The first functional component can be a moiety capable of reacting with an amino acid. The moiety can be, for example, isothiocyanate (e.g., PITC), dinitrofluorobenzene, dansyl chloride, or other amino acid reactive groups. In some cases, the second functional component can comprise a reactive group capable of coupling to a polymerizable molecule, which can be a nucleic acid molecule (e.g., DNA, RNA, LNA, PNA), a peptide, or a synthetic organic compound. In the case of a DNA-based polymerizable group, the DNA can comprise primer sequences, enzyme ligation and chemical conjugation sites, barcode sequences, or combinations thereof. In some cases, the linker can be pre-functionalized to contain a polymerizable molecule (e.g., as shown in Figure 4 Partition B), as follows:

[0254] Method 1: Preparation of linker-DNA sequence conjugate

[0255] Synthesize a DNA sequence of 15 - 150 bp in length with an internal azide modification (e.g., azide-dT modification, Integrated DNA Technologies) or an external azide modification at the 5' or 3' end. A linker comprising (i) a PITC moiety (amino acid reactive group) and (ii) an alkyne moiety can be conjugated to the DNA sequence by copper-catalyzed click chemistry as follows: Prepare 100 μM linker and 10 μM of the DNA sequence in a reaction buffer consisting of 200 μM tris((1-benzyl-1H-1,2,3-triazol-4-yl)methyl)amine (TBTA), 500 μM TCEP, and 200 μM CuSO 4 4, dissolved in 1x PBS, pH 7.86. The reaction is incubated at room temperature for 2 hours. The linker-DNA sequence conjugate is then purified from the reaction by reverse-phase high performance liquid chromatography (HPLC).

[0256] Method 2: Preparation of linker-peptide conjugate

[0257] Peptide sequences with internal cysteine residues (ranging in length from 10 - 50 aa) can be synthesized. The peptide can be alkylated with iodoacetamide azide as follows: The peptide is reduced by preparing it to 1 mg / ml in a solution containing 1% SDS and 100 mM ammonium bicarbonate (pH 8.0). Tris(2 - carboxyethyl)phosphine hydrochloride (TCEP - HCl) is added to this solution to a final concentration of 10 mM and incubated at 55 degrees Celsius for 1 hour. Subsequently, iodoacetamide azide is added to a final concentration of 10 mM and incubated at room temperature for 1 hour. Then the alkylated peptide is purified by buffer exchange twice using a buffer exchange column. Then, the alkylated peptide reacts with a linker containing (i) a PITC moiety (amino acid - reactive group) and (ii) an alkyne moiety via copper - catalyzed click chemical reaction. The linker (linker - peptide sequence conjugate) is prepared at 100 μM and the alkylated peptide sequence at 10 μM in a reaction buffer consisting of 200 μM tris((1 - benzyl - 1H - 1,2,3 - triazol - 4 - yl)methyl)amine (TBTA), 500 μM TCEP, and 200 μM CuSO 4 and dissolved in 1x PBS, pH 7.86. The reaction is incubated at room temperature for 2 hours. Then the linker (linker - peptide sequence conjugate) is purified from the reaction using reverse - phase high - performance liquid chromatography (HPLC).

[0258] Example 2 - Detection of Amino Acids Conjugated with Polymerizable Molecules by Nanopore

[0259] To determine whether it is possible to detect modified amino acids or their derivatives, such as AALC complexes or stacked AALC complexes or parts thereof, via nanopore, an amino acid - linker - polymerizable molecule complex is generated and translocated through a nanopore sequencing system. The amino acid - linker - polymerizable molecule complex is generated by coupling a polymerizable molecule with an amino acid - linker complex. The polymerizable molecule includes an alkyne - linked DNA molecule. More specifically, the polymerizable molecule includes an alkyne linker located at position T236 within a 16 - nt poly - dT DNA oligomer scaffold in the middle of one strand of a 450 - nt dsDNA backbone, as schematically shown in Figure 6A . The amino acid - linker complex is generated using the bifunctional linker 1 - (2 - azidoethyl)-4 - isothiocyanatobenzene, which contains (1) a PITC moiety (amino acid - reactive group) and (2) an azide moiety capable of reacting with the alkyne of the polymerizable molecule. The bifunctional linker reacts via the PITC moiety with three different amino acids: Asp, Trp, and Tyr, thereby generating the amino acid - linker complex, which is also shown in the bottom partition of Figure 6A . Then, the amino acid - linker complex reacts with the alkyne - linked DNA molecule, thereby generating an amino acid - linker - polymerizable molecule conjugate.

[0260] Next, the amino acid-linker-polymerizable molecule conjugates were run through a nanopore sequencing system (Oxford Nanopore Technologies MinION) to see if the resulting current signals could be assigned back to the amino acid type. The unmodified DNA backbone (“unmodified”) was used as a baseline control, and the alkyne-linked DNA molecule (“alkyne”) was used as a control for alkyne linker-specific current blockade. Figure 6B The current traces and assigned nucleotide sequences of the DNA backbones are shown. Notably, all different molecules pass through the nanopore, and discernible qualitative differences or perturbations in the current signals can be seen from the amino acid-linker-polymerizable molecule conjugates (labeled “linker-Asp”, “linker-Trp”, or “linker-Tyr”), which are not present in the current signals of the control molecules. Figure 6C The variation of the current traces (measured current signals) with time under five conditions is shown: unmodified, alkyne, linker-Asp, linker-Trp, and linker-Tyr. The left shows the combined superimposed traces of all reads of the nanopore in the device, and the right shows three representative individual traces. Figure 6D The variation of the current traces (measured current signals) of the five samples with base pair (nucleotide) position is shown: unmodified, alkyne, linker-Asp, linker-Trp, and linker-Tyr. The left shows the combined superimposed traces of all reads of the nanopore in the device, and the middle shows the average signal, and the error bars represent the standard deviation. The right radial plot represents the base or nucleotide position (along the radial axis) and the measured median current for each of the five test conditions. Interestingly, the presence of the amino acid-linker complex on the polymerizable molecule affects the current signals 2 nt before and 5 nt after the conjugation site, suggesting that to minimize interference from neighboring amino acids, the polymerizable molecule spacer of the amino acid-linker should be >8 nt. Next, to determine how the current traces of the amino acid-linker-polymerizable molecule complex differ from the control molecules, a custom machine learning algorithm model was used. Figure 6E The normalized confusion matrix is shown, where the x-axis is the classified molecule (unmodified, alkyne, linker-Asp, linker-Trp, and linker-Tyr), and the y-axis is the actual molecule. As can be seen from the figure, the classification of the correct molecule is achieved with moderate accuracy.

[0261] Example 3 - Multiplexed Readout of Amino Acids Conjugated to Barcoded Polymerizable Molecules

[0262] To determine whether it is possible to multiplex information for nanopore detection (e.g., of modified amino acids or their derivatives containing barcoded polymerizable molecules such as nucleic acid barcoded molecules), amino acid-linker-barcoded polymerizable molecule complexes were generated in a method similar to that described above. The barcoded polymerizable molecule contains a 4-nt barcode sequence located 5’ of an alkyne linker that is positioned at the T236 position within a 450 nt ssDNA backbone, as schematically shown in Figure 7A . Two types of ssDNA backbones were tested: the first contains a poly-dT region, as described above and schematically shown in Figure 6A , and the second contains a mixed base region, as shown in Figure 7A . Next, amino acid-linker complexes were generated using the bifunctional linker 1-(2-azidoethyl)-4-isothiocyanatobenzene, which contains (1) a PITC moiety (an amino acid-reactive group) and (2) an azide moiety capable of reacting with the alkyne of the polymerizable molecule. The bifunctional linker reacts through the PITC moiety with two different amino acids: Asp and Tyr, thereby generating amino acid-linker complexes (identical to those shown in Figure 6A ). The amino acid-linker complexes were then reacted with the alkyne-linked DNA molecules to generate amino acid-linker-polymerizable molecule complexes.

[0263] Next, the amino acid-linker-polymerizable molecule complexes were run through a nanopore sequencing system (Oxford Nanopore Technologies MinION) to see whether the resulting current signals could be assigned back to the amino acid type (Asp or Tyr), and also whether the barcode sequences of the barcoded polymerizable molecules were distinguishable. Alkyne-linked DNA molecules without the amino acid-linker complex (“alkyne”) were used as a control for alkyne linker-specific current blockage. Figure 7B Shows the variation of the current traces (measured current signals) of the amino acid-linker-polymerizable molecule complexes of barcoded polymerizable molecules containing poly-dT sequences (“poly-dT backbone”) and mixed base sequences (“mixed base backbone”) over time (ms). For both types of polymerizable molecules, distinguishable differences or perturbations in the current signals could be seen from the amino acid-linker-polymerizable molecule conjugates (“linker-Asp” and “linker-Tyr”). Figure 7CShows the current traces (measured current signals) of two types of polymerizable molecules as a function of base (nucleotide) position under three conditions: linker-Tyr, linker-Asp, and alkyne (control). Interestingly, compared to poly-dT polymerizable molecules, the mixed-base polymerizable molecules produce more distinguishable current traces, indicating that using mixed-base polymerizable molecules instead of repetitive nucleotide sequences can generate more distinguishable current traces. These results also suggest that multiplexed readout of both DNA sequence and amino acid type is possible.

[0264] Example 4 - Effect of Linker Length of Polymerizable Molecules on Amino Acid Nanopore Current Readout Conjugated to Polymerizable Molecules

[0265] As described herein, polymerizable molecules of modified amino acids or their derivatives (e.g., AALC complexes or stacked AALC complexes or portions thereof) may contain a linker containing a click chemistry moiety that can be coupled to an amino acid-linker complex or another linker containing (i) a complementary click chemistry moiety and (ii) an amino acid reactive group. To determine whether the intramolecular distance (i.e., linker length) between the click chemistry moiety and the polymerizable molecule can affect nanopore detection of modified amino acids or their derivatives (e.g., amino acid-linker-polymerizable complexes), two different amino acid-linker-polymerizable complexes were generated. The first amino acid-linker-polymerizable complex contains an octadiynyl dU linker, as Figure 7A shown, and the second amino acid-linker-polymerizable complex contains an ethynyl dU linker, as Figure 8A shown. Both of these amino acid-linker-polymerizable complexes contain the same DNA backbone, which includes a 4-nt barcode sequence located 5’ of the modified nucleotide (octadiynyl dU or ethynyl dU), and the modified nucleotide is located at the 199 nt position within a 400 nt DNA backbone. An amino acid-linker complex was generated using the bifunctional linker 1-(2-azidoethyl)-4-isothiocyanatobenzene, which contains (1) a PITC moiety (amino acid reactive group) and (2) an azide moiety capable of reacting with the alkyne of the polymerizable molecule. The bifunctional linker reacts with two different amino acids: Asp and Tyr through the PITC moiety, thereby generating amino acid-linker complexes (the same as those shown in Figure 6A ). Then, the amino acid-linker complexes react with the alkyne-linked DNA molecules through copper-catalyzed click chemistry, thereby generating amino acid-linker-polymerizable molecule complexes.

[0266] Figure 8BShows the current traces (measured current signal minus background) of amino acid-linker-polymerizable molecule complexes containing ethynyl dU linkers as a function of time (top) or base pair (nucleotide) position (bottom) for five different molecules: a control polymerizable molecule with an ethynyl dU linker but no amino acid-linker complex (“alkyne”), and amino acid-linker-polymerizable molecule complexes including linker-Glu, linker-Phe, linker-Gly, and linker-Trp. The current traces of the complexes containing amino acids (linker-Glu, linker-Phe, linker-Gly, and linker-Trp) show distinct differences compared to the control molecules without amino acid-linker complexes.

[0267] Figure 8C Shows the current traces (measured current signal) of amino acid-linker-polymerizable molecule complexes containing ethynyl dU linkers (top) or octadiynyl dU linkers (bottom) as a function of time for four or five different molecules: a control polymerizable molecule with an ethynyl dU linker or octadiynyl dU but no amino acid-linker complex (“alkyne”), and amino acid-linker-polymerizable molecule complexes including linker-Glu, linker-Phe, linker-Gly, linker-Trp (for ethynyl dU linkers) or linker-Asp, linker-Tyr, or linker-Trp (for octadiynyl dU linkers). The current traces appear to indicate that the longer linker size (octadiynyl dU) creates a greater contrast between the modified molecules (amino acid-linker-polymerizable molecule complexes) and the unmodified control molecules (polymerizable molecules not conjugated with amino acid-linker complexes).

[0268] Figure 8D Shows a t-SNE plot of the dynamic time warping (DTW) correlation matrix comparing the nanopore current traces of amino acid-linker-polymerizable molecule complexes containing ethynyl dU linkers (left) or octadiynyl dU (right) linkers to control polymerizable molecules with ethynyl dU linkers or octadiynyl dU linkers but no amino acid-linker complexes (“alkynes”). Amino acid-linker-polymerizable molecule conjugates of linker-Trp (“W”) are shown. The t-SNE plot indicates that the longer linker size (octadiynyl dU) creates a greater contrast between the modified (amino acid-linker-polymerizable molecule complexes, “W”) and unmodified control molecules (polymerizable molecules not conjugated with amino acid-linker complexes, “alkynes”).

[0269] Example 5 - Effect of Modified Amino Acids or Their Derivatives on Translocation Velocity

[0270] To determine the effect of a modified amino acid or its derivative (e.g., an amino acid comprising a polymerizable molecule, an AALC complex, a stacked AALC complex, or a portion thereof) on nanopore sequencer readout, the dwell time was measured. The dwell time of a base is the duration of time that an individual base resides in the nanopore and corresponds to the translocation speed of the molecule through the nanopore. Several amino acid-linker-barcoded polymerizable molecule conjugates were measured. Amino acid-linker-polymerizable molecules were synthesized as described above using an octadiynyl dU linker on the polymerizable molecule as schematically illustrated in Figure 6A above. The amino acid-linker-polymerizable molecule conjugates were run through a nanopore sequencing system (Oxford Nanopore Technologies MinION) to see if modification of the polymerizable molecule (e.g., addition of an amino acid-linker complex) affects the translocation speed. An unmodified DNA backbone (“unmodified”) was used as a baseline control, and an alkyne-linked DNA molecule (“alkyne”) was used as a control for alkyne linker-specific current blocking. Figure 9 A heat map of the measured dwell times of the control molecule and three different amino acid-linker-polymerizable molecule complexes (labeled “linker-Asp,” “linker-Trp,” or “linker-Tyr”) is shown. The x-axis represents the base pair position, which is truncated to show a 25-nt region that encompasses and flanks a poly-dT sequence, where the alkyne linker is present at the 13-nt position. The y-axis represents each individual current trace. The relative intensity at each time point represents the relative dwell time at that time point (each time point corresponds to 0.25 milliseconds). Notably, the measured dwell times of the amino acid-linker-polymerizable molecule complexes were different compared to the control molecule. Interestingly, the modification (addition of an alkyne linker or an alkyne linker conjugated to an amino acid-linker complex) appears to affect the dwell time of the DNA backbone at multiple positions, including base pair positions 0-5 and 10-25 flanking the modification site, suggesting that some molecular stretching and contraction can occur and that the presence of the AALC can affect a substantial nucleotide window around the modification site.

[0271] Example 6 - Nanopore Sequencing of Stacked Amino Acid Polymerizable Complexes

[0272] To determine if a modified amino acid or its derivative (e.g., a stacked AALC complex or a portion thereof) can be detected by nanopore, an amino acid-linker-polymerizable molecule conjugate containing two amino acids was first generated as schematically shown in Figure 10A above. The polymerizable molecule contains alkyne linkers (octadiynyl dU) at two positions within a 252-nt DNA backbone as Figure 10BAs shown. Next, a bifunctional linker, 1-(2-azidoethyl)-4-isothiocyanatobenzene, is used to generate an amino acid-linker complex. The linker contains (1) a PITC moiety (amino acid reactive group) and (2) an azide moiety capable of reacting with the alkyne of the polymerizable molecule. The bifunctional linker reacts with three different amino acids, Asp, Trp, and Tyr, through the PITC moiety to generate the amino acid-linker complex, which is also shown in Figure 6A the bottom partition of. Then, the amino acid-linker complex reacts with the alkyne-linked DNA molecule to generate a bis-amino acid-linker-polymerizable molecule complex containing a pair of amino acids. Figure 10C A table showing different pairs of amino acids conjugated at the first or second linker position is presented. The unmodified DNA backbone ("unmodified") is used as a baseline control, while the alkyne-linked DNA molecule ("alkyne") is used as a control for alkyne linker-specific current blocking.

[0273] Next, the bis-amino acid-linker-polymerizable molecule conjugate is run through a nanopore sequencing system (Oxford Nanopore Technologies MinION) to see if the resulting current signal can be assigned back to the two types of amino acids present in the bis-amino acid-linker-polymerizable molecule complex. Figure 10D The current trace (measured current signal) as a function of time (top) or base pair position (bottom) is shown. Similar to the amino acid-linker-polymerizable molecule complex containing a single amino acid, the bis-amino acid-linker-polymerizable molecule complex containing two amino acids passes through the nanopore, and distinguishable differences or perturbations can be seen in the current signal for each amino acid in the bis-amino acid-linker-polymerizable molecule conjugate (labeled "Asp / Trp", "Asp / Tyr", or "Trp / Tyr") at their respective positions (indicated by arrows), which are not present in the current signal of the control molecule. Next, to determine the difference in the current trace of the bis-amino acid-linker-polymerizable molecule complex containing two amino acids from the control molecule, a custom machine learning algorithm model is used. Figure 10E The normalized confusion matrix of the predicted molecules (unmodified, alkyne, Asp / Trp, Asp / Tyr, Trp / Tyr) on the x-axis and the actual molecules on the y-axis is shown. It can be seen from the figure that the classification of the correct molecules has a high accuracy, ranging from 92% to 98%. In summary, these results suggest that processing amino acids to generate modified amino acids containing polymerizable molecules can be a viable method for high-precision peptide sequencing using nanopore or nanochannel methods.

[0274] Example 7 - Temperature Regulation to Improve the Current Signal-to-Noise Ratio

[0275] As described herein, the translocation rate of a modified amino acid or its derivative can be different from that of an unmodified amino acid. Similarly, the translocation rate of a modified amino acid or its derivative containing a polymerizable molecule can be different from that of a polymerizable molecule without a conjugated amino acid. In one hypothesis, the translocation rate of a molecule through a nanopore can be decreased by lowering the ambient temperature. To test this hypothesis, an unmodified polymerizable molecule (not containing an amino acid-linker complex) can be passed through a nanopore sequencing system at ambient temperature (room temperature) or on ice.

[0276] The polymerizable molecule includes a 450 nt dsDNA backbone that contains a 16 nt poly-dT sequence, as Figure 11A schematically shown in Figure 11A (showing the selected sequence from approximately ~225 bp to ~245 bp). The polymerizable molecule is run through a nanopore sequencing system (Oxford Nanopore Technologies MinION) to see the effect of temperature on the sequencing output. The measured temperature of the flow cell run at room temperature is 34 degrees Celsius, and the measured temperature of the flow cell run on ice is ~19 - 20 degrees Celsius.

[0277] Figure 11B Shows a histogram of the translocation duration, which is a measure of the length of time that a region or the entire polymerizable molecule resides in the nanopore, corresponding to the translocation rate of the region or the entire polymerizable molecule. The translocation duration of the polymerizable molecule is measured in milliseconds (ms) under two different temperature conditions (room temperature or ice). The left plot shows the translocation duration of the entire polymerizable molecule, and the right plot shows the translocation duration at selected sites around the 16 nt poly-dT sequence (positions 200 - 275 nt). As can be seen from the histogram, the ice condition results in a decrease in the translocation rate (an increase in the translocation duration), with a greater variability or range in the measured duration values.

[0278] Figure 11CShows a histogram of the translocation duration (in milliseconds) of polymerizable molecules under two different temperature conditions (room temperature or ice). The left plot shows the translocation duration of polymerizable molecules at positions 200 - 225 nt upstream of the 16 nt poly-dT sequence. The middle plot shows the translocation duration at selected sites immediately adjacent to and including the 16 nt poly-dT sequence (positions 225 - 250 nt). The right plot shows the translocation duration of polymerizable molecules at positions 250 - 275 nt downstream of the 16 nt poly-dT sequence. As can be seen from all three plots, the ice condition results in a decrease in the translocation speed (an increase in the translocation duration), and there is a greater variability or range in the measured duration values for the poly-dT region and adjacent regions.

[0279] Figure 11D Shows the variation of the current trace (measured current signal) starting from positions 200 - 280 nt with base position. The left plot shows the current trace under room temperature conditions, and the right plot shows the current trace under ice conditions. The ice condition improves the signal-to-noise ratio of the current, as demonstrated by the decrease in the magnitude of the error bars in the poly-dT region (∼225 - 245 nt region).

[0280] Figure 11E Shows the variation of the translocation duration (in milliseconds) starting from positions 200 - 280 nt with base position. The left plot shows the current trace under room temperature conditions, and the right plot shows the current trace under ice conditions. Interestingly, while the ice condition results in an increase in the dwell time at each base position, it also leads to an increase in the noise of the translocation duration outside the poly-dT region (∼225 - 245 bp region).

[0281] In summary, Figures 11A-11E the results indicate that decreasing the temperature can improve the signal-to-noise ratio of the current trace of polymerizable molecules read out from a nanopore sequencing system, which can be applicable to improving the readout accuracy of modified amino acids or their derivatives.

[0282] Although the preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. The present invention is not intended to be limited by the specific embodiments provided within this specification. While the present invention has been described with reference to the foregoing specific description, the description and illustration of the embodiments herein are not intended to be construed in a limiting sense. Various variations, changes, and substitutions will now occur to those skilled in the art without departing from the present invention. In addition, it should be understood that all aspects of the present invention are not limited to the specific depictions, configurations, or relative proportions described herein, which depend on various conditions and variables. It should be understood that various alternatives to the embodiments of the present invention described herein may be used to practice the present invention. Accordingly, it is contemplated that the present invention should also cover any such alternatives, modifications, variations, or equivalents. The following claims are intended to define the scope of the present invention and thereby cover the methods and structures within the scope of these claims and their equivalents.

Claims

1. A method for processing a modified amino acid, the method comprising: (a) providing the modified amino acid, wherein the modified amino acid comprises a polymerizable molecule; and (b) translocating the modified amino acid or a derivative thereof through a nanopore or a nanogap; wherein the polymerizable molecule causes a change in the translocation rate of the modified amino acid or a derivative thereof through the nanopore or the nanogap as compared to the translocation rate of an amino acid without the polymerizable molecule.

2. A method, comprising: (a) providing a modified amino acid, wherein the modified amino acid comprises a polymerizable molecule; and (b) translocating the modified amino acid or a derivative thereof through a nanopore or a nanogap; (c) measuring a signal from the nanopore or the nanogap; wherein the polymerizable molecule causes an increase in the signal-to-noise ratio (SNR) of the signal in (c) as compared to the measured SNR of an amino acid without the polymerizable molecule.

3. The method according to claim 1 or 2, wherein the modified amino acid comprises or is derived from a proteinogenic amino acid conjugated to the polymerizable molecule.

4. The method according to any one of claims 1-3, wherein the modified amino acid or a derivative thereof further comprises a binder.

5. The method according to claim 4, wherein the binder comprises an antibody, an antibody fragment, a nanobody, an aptamer, a peptide, a polymer, an inorganic compound, a small molecule, or a derivative thereof.

6. The method according to any one of claims 1-5, wherein the modified amino acid comprises a non-naturally occurring chemical modification.

7. The method according to claim 6, wherein the non-naturally occurring chemical modification is a protecting group.

8. The method according to any one of claims 1-7, wherein the nanopore is conjugated to a helicase.

9. The method according to claim 8, wherein the helicase guides the modified amino acid or a derivative thereof through the nanopore.

10. The method according to any one of claims 1-9, wherein (b) is performed using a topoisomerase or a polymerase.

11. The method according to any one of claims 1-9, wherein (b) is performed using an electric field or a magnetic field.

12. The method according to any one of claims 1-11, wherein the polymerizable molecule comprises a nucleic acid molecule.

13. The method according to claim 12, wherein the nucleic acid molecule comprises a deoxyribonucleic acid (DNA) molecule, a xeno nucleic acid (XNA) molecule, a ribonucleic acid (RNA) molecule, or a modified variant thereof.

14. The method according to any one of claims 1-13, wherein the nanopore comprises a transmembrane protein.

15. The method according to any one of claims 1-13, wherein the nanogap comprises an inorganic material.

16. The method according to claim 15, wherein the nanogap comprises silicon nitride or molybdenum sulfate.

17. The method according to any one of claims 1-16, wherein the polymerizable molecule is covalently linked to the modified amino acid.

18. The method according to any one of claims 1-17 further comprises generating the modified amino acid from an amino acid of the peptide before (a).

19. The method according to claim 18, wherein the amino acid is at the end of the peptide.

20. The method according to claim 19, wherein the end is the N-terminus.

21. The method according to claim 19, wherein the modified amino acid comprises the polymerizable molecule linked to the terminal amino acid.

22. The method according to claim 21, wherein the polymerizable molecule is linked to the terminal amino acid via an isothiocyanate moiety.

23. The method according to any one of claims 1-22 further comprises, before (a), (i) providing a linker, wherein the linker comprises an amino acid-reactive moiety, and (ii) contacting the amino acid-reactive moiety with a protein native amino acid to produce the modified amino acid.

24. The method according to claim 23, wherein the linker is coupled to the polymerizable molecule.

25. The method according to claim 23 further comprises coupling the linker to the polymerizable molecule.

26. The method according to claim 25, wherein the linker comprises a first reactive moiety and the polymerizable molecule comprises a second reactive moiety, and further comprises reacting the first reactive moiety with the second reactive moiety to generate the linker coupled to the polymerizable molecule.

27. The method according to claim 26, wherein the first reactive moiety or the second reactive moiety comprises a click chemistry moiety.

28. The method according to claim 26, wherein the polymerizable molecule comprises a nucleic acid linker, wherein the nucleic acid linker comprises a modified nucleobase and a click chemistry moiety.

29. The method according to any one of claims 23-27, wherein the amino acid-reactive moiety comprises phenyl isothiocyanate (PITC).

30. The method according to any one of claims 23-29, wherein the protein native amino acid is the terminal amino acid of the peptide.

31. The method according to claim 30 further comprises tethering the modified amino acid to a capture moiety and cleaving the terminal amino acid from the peptide to produce a remaining peptide and an amino acid-linker-capture moiety (AALC) complex.

32. The method according to claim 31, wherein the capture moiety is coupled to a substrate.

33. The method according to claim 31, wherein the capture moiety comprises a first nucleic acid molecule and wherein the polymerizable molecule comprises a second nucleic acid molecule.

34. The method according to claim 33, wherein the first nucleic acid molecule or the second nucleic acid molecule comprises a nucleic acid barcode molecule.

35. The method according to claim 34, wherein the nucleic acid barcode molecule identifies the peptide.

36. The method according to any one of claims 33-35, wherein the nucleic acid barcode molecule contains time information.

37. The method according to any one of claims 31 - 36, wherein the capture moiety comprises a cleavable moiety.

38. The method according to any one of claims 33 - 36, wherein the tethering comprises linking the first nucleic acid molecule to the second nucleic acid molecule.

39. The method according to claim 38, wherein the linking is performed using a ligase.

40. The method according to claim 38, wherein the linking comprises (i) providing a splint oligonucleotide comprising a first sequence complementary to at least a portion of the first nucleic acid molecule and a second sequence complementary to at least a portion of the second nucleic acid molecule, and (ii) hybridizing the first sequence to the portion of the first nucleic acid molecule and hybridizing the second sequence to the portion of the second nucleic acid molecule.

41. The method according to claim 33, further comprising: (I) providing an additional linker conjugated to an additional nucleic acid molecule, (II) contacting the additional linker with the terminal amino acid of the remaining peptide to produce an additional modified amino acid; and (III) tethering the additional modified amino acid to the AALC complex to produce the derivative thereof.

42. The method according to claim 41, further comprising: repeating (I) - (III) to produce the derivative thereof, wherein the derivative thereof comprises a plurality of modified amino acids.

43. The method according to claim 41, wherein the nucleic acid molecule or the additional nucleic acid molecule comprises a barcode sequence.

44. The method according to any one of claims 41 - 43, wherein the nucleic acid molecule comprises a first sequence, wherein the additional nucleic acid molecule comprises a second sequence, wherein the first sequence is different from the second sequence.

45. The method according to claim 44, wherein the first sequence or the second sequence comprises time or spatial information.

46. The method according to claim 45, wherein the time information is an index of the number of cycles or iterations of providing the linker or the additional linker.

47. The method according to claim 1, wherein the translocation rate of the modified amino acid is lower compared to the translocation rate of the amino acid without the polymerizable molecule.

48. The method according to claim 1, further comprising measuring the current signal of the nanopore or the nanogap to determine the translocation rate.

49. The method according to any one of claims 1 - 48, further comprising identifying the modified amino acid or its derivative using the nanopore or the nanogap.

50. The method according to claim 49, wherein the identification comprises measuring the current signal from the nanopore or the nanogap and determining the amino acid type of the modified amino acid or its derivative or its post - translational modification variant.

51. The method according to claim 1 or 2, wherein (b) occurs at a temperature below ambient temperature.

52. A method of processing a peptide, comprising: (a) Provide the peptide and the linker, wherein the linker is capable of coupling to an amino acid of the peptide; (b) Couple the linker to the amino acid of the peptide; (c) Couple the linker to a capture moiety; (d) Cleave the amino acid from the peptide to produce an amino acid-linker-capture moiety (AALC) complex; (e) Provide an additional linker, wherein the additional linker is capable of coupling to another amino acid of the peptide; (f) Couple the additional linker to the another amino acid, thereby producing an additional amino acid-linker complex; and (g) Couple the additional amino acid-linker complex to the AALC complex, thereby producing a stacked AALC complex; wherein the coupling in (c) is carried out using a polymerizable molecule.

53. The method according to claim 52, wherein the linker comprises a reactive moiety.

54. The method according to claim 53, wherein the polymerizable molecule comprises an additional reactive moiety capable of reacting with the reactive moiety of the linker, and wherein, prior to (a), the polymerizable molecule is coupled to the linker by reacting the reactive moiety with the additional reactive moiety.

55. The method according to claim 54, wherein the polymerizable molecule comprises an additional linker, the additional linker comprises the additional reactive moiety, wherein the additional linker comprises a modified nucleobase, and the additional reactive moiety comprises a click chemistry moiety.

56. The method according to claim 53, further comprising providing the polymerizable molecule prior to (c).

57. The method according to any one of claims 52-56, wherein the capture moiety is immobilized on a substrate.

58. The method according to claim 57, wherein the substrate is substantially planar.

59. The method according to claim 57, wherein the substrate is a bead.

60. The method according to any one of claims 52-59, further comprising providing the capture moiety, wherein the capture moiety comprises a nucleic acid molecule.

61. The method according to claim 60, wherein the nucleic acid molecule comprises a DNA molecule, an RNA molecule, an XNA molecule, or a modified variant thereof.

62. The method according to claim 61, wherein the DNA molecule is single-stranded.

63. The method according to claim 61, wherein the polymerizable molecule comprises an additional nucleic acid molecule.

64. The method according to claim 63, wherein (c) comprises coupling the additional nucleic acid molecule to the nucleic acid molecule of the capture moiety.

65. The method according to claim 64, wherein the coupling is carried out using a ligase.

66. The method according to claim 64, wherein at least a portion of the additional nucleic acid molecule is complementary to at least a portion of the nucleic acid molecule of the capture moiety.

67. The method according to claim 66, wherein the coupling is carried out by hybridizing at least a portion of the additional nucleic acid molecule to at least a portion of the nucleic acid molecule.

68. The method according to claim 64, wherein the additional nucleic acid molecule is coupled to the nucleic acid molecule of the capture moiety using a splint oligonucleotide.

69. The method according to any one of claims 60-68, wherein the capture moiety comprises a nucleic acid barcode molecule.

70. The method according to claim 69, wherein the nucleic acid barcode molecule identifies the peptide.

71. The method according to any one of claims 60-70, wherein the capture moiety is coupled to the peptide.

72. The method according to any one of claims 52-71, wherein the amino acid or the other amino acid is an N-terminal amino acid or a C-terminal amino acid.

73. The method according to any one of claims 52-72, wherein (b) occurs before (c).

74. The method according to any one of claims 52-72, wherein (c) occurs before (b).

75. The method according to any one of claims 52-74, further comprising repeating (a)-(g).

76. The method according to any one of claims 52-75, further comprising identifying the AALC complex or the stacked AALC complex.

77. The method according to claim 76, wherein the identification comprises using a nanopore or a nanogap.

78. The method according to claim 77, wherein the identification of the translocation of the AALC complex or the stacked AALC complex comprises translocating the AALC complex or the stacked AALC complex or a part thereof through the nanopore or the nanogap.

79. The method according to claim 78, wherein the translocation occurs at a temperature below ambient temperature.

80. The method according to claim 76, wherein identifying the AALC complex or the stacked AALC complex comprises determining the amino acid type of the AALC complex or the amino acid type of the stacked AALC complex.

81. The method according to any one of claims 52-80, wherein the polymerizable molecule comprises a nucleic acid barcode molecule containing time information.

82. The method according to any one of claims 52-81, wherein the capture moiety comprises a cleavable moiety.

83. A method comprising: (a) providing a nucleic acid molecule coupled to an amino acid or a modified amino acid; (b) sequencing the nucleic acid molecule; and (c) identifying the amino acid or the modified amino acid; wherein (b) and (c) are completed within 1 minute of each other.

84. The method according to claim 83, wherein (b) comprises generating a sequencing read.

85. The method according to any one of claims 83-84, wherein (b) or (c) is performed using a nanopore.

86. The method according to claim 85, wherein the nanopore is coupled to a helicase, a topoisomerase, an unfoldase, or a polymerase.

87. The method according to any one of claims 83-86, wherein (b) and (c) are performed using a nanopore.

88. The method according to any one of claims 83 - 87, wherein the nucleic acid molecule is conjugated to the amino acid or the modified amino acid via a linker.

89. The method according to claim 88, wherein, prior to (a), the linker comprises an amino acid - reactive moiety and is conjugated to the nucleic acid molecule.

90. The method according to claim 89, wherein, prior to (a), the linker comprises an additional reactive moiety.

91. The method according to claim 88, wherein the modified amino acid comprises a protein - native amino acid, the linker, and the nucleic acid molecule.

92. The method according to claim 88, wherein the linker comprises at least 2 atoms.

93. The method according to claim 88, wherein the linker comprises a phenyl isothiocyanate moiety.

94. The method according to claim 88, further comprising, prior to (a), conjugating the linker to the amino acid or the modified amino acid to generate an amino acid - linker complex conjugated to the nucleic acid molecule.

95. The method according to claim 94, further comprising, prior to (b) or (c), conjugating the amino acid - linker complex to a capture moiety, thereby generating an amino acid - linker - capture moiety (AALC) complex.

96. The method according to claim 95, wherein the capture moiety comprises a nucleic acid molecule.

97. The method according to claim 95, wherein, prior to (a), the amino acid or the modified amino acid is conjugated to a peptide, and further comprising cleaving the AALC complex from the peptide.

98. The method according to claim 97, wherein the capture moiety comprises a nucleic acid barcode molecule that recognizes the peptide.

99. The method according to claim 97, further comprising providing an additional linker, conjugating the additional linker to an additional amino acid of the peptide to generate an additional amino acid - linker complex, and conjugating the additional amino acid - linker complex to the AALC complex, thereby generating a stacked AALC complex.

100. The method according to claim 95, wherein the capture moiety comprises a cleavable moiety.

101. The method according to any one of claims 91 - 99, wherein the nucleic acid molecule comprises time information.

102. The method according to claim 83, wherein the identification comprises measuring a current signal from the amino acid or the modified amino acid using a nanopore or a nanogap and determining the amino acid type of the amino acid or the modified amino acid or a post - translational modification variant thereof.

103. The method according to claim 102, wherein the measurement occurs at a temperature below ambient temperature.

104. The method according to claim 83, wherein the nucleic acid molecule comprises a modified nucleobase and a click - chemistry moiety.

105. A composition, comprising: a linker covalently attached to a nucleic acid barcode molecule, wherein the linker comprises an amino acid - reactive group, and wherein the nucleic acid barcode molecule comprises time information.

106. The composition according to claim 105, wherein the amino acid reactive group comprises isothiocyanate.

107. The composition according to claim 106, wherein the isothiocyanate is PITC.

108. The composition according to claim 105, wherein the nucleic acid barcode molecule comprises a modified nucleobase and a click chemistry moiety.

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