Macromolecule analysis with nucleic acid coding

Abstract

Methods of analyzing macromolecules, including peptides, polypeptides, and proteins, using nucleotide coding are disclosed.

Description

[0001] This case is a divisional application of a case with an application date of May 2, 2017, an invention name of “Macromolecular Analysis Using Nucleic Acid Encoding” and an application number of 201780041157.0.

[0002] Sequence Listing Statement

[0003] The sequence listing associated with this application is provided in text format in lieu of a paper copy and is incorporated into this specification by reference. The name of the text file containing the sequence listing is 760229_401WO_SEQUENCE_LISTING.txt. The text file is 38.7KB, created on May 2, 2017, and submitted electronically via EFS-Web. Technical Field

[0004] The present disclosure generally relates to the analysis of macromolecules, including peptides, polypeptides, and proteins, using barcodes and nucleic acid encoding of molecular recognition events.

[0005] Related Technical Notes Coding Tags

[0006] Proteins play an integral role in cell biology and physiology, performing and facilitating many different biological functions. The collection of different protein molecules is very rich and much more complex than the transcriptome due to the additional diversity introduced by post-translational modifications (PTMs). In addition, proteins within cells change dynamically (in expression levels and modification states) in response to the environment, physiological state, and disease state. Therefore, proteins contain a wealth of unexplored relevant information, especially related to genomic information. In general, innovation in proteomic analysis has lagged relative to genomic analysis. In the field of genomics, next-generation sequencing (NGS) has transformed the field by analyzing billions of DNA sequences in a single instrument run, while in protein analysis and peptide sequencing, throughput remains limited.

[0007] However, this protein information is urgently needed to better understand proteome dynamics in health and disease and to help achieve precision medicine. Therefore, there is great interest in developing "next generation" tools to miniaturize and highly parallelize the collection of this proteomic information.

[0008] Highly parallel macromolecular characterization and protein identification are challenging for a variety of reasons. Using affinity-based assays is often difficult due to several key challenges. An obvious challenge is multiplexing the readouts of a set of affinity reagents to a set of homologous macromolecules; another challenge is minimizing cross-reactions between affinity reagents and off-target macromolecules; and a third challenge is developing efficient high-throughput readout platforms. An example of this problem occurs in proteomics, where one of the goals is to identify and quantify most or all proteins in a sample. In addition, it is desirable to characterize various post-translational modifications (PTMs) on proteins at the single-molecule level. Currently, this is a daunting task to accomplish in a high-throughput manner.

[0009] Molecular identification and characterization of protein or peptide macromolecules are often performed using immunoassays. There are many different immunoassay formats, including ELISA, multiplexed ELISA (e.g., spotted antibody arrays, liquid particle ELISA arrays), digital ELISA (e.g., Quanterix, Singulex), reverse phase protein arrays (RPPA), and the like. These different immunoassay platforms all face similar challenges, including the development of high affinity and high specificity (or selectivity) antibodies (binders), limited ability to multiplex at the sample and analyte levels, limited sensitivity and dynamic range, as well as cross-reactivity and background signals. Binder-agnostic methods, such as direct protein characterization by peptide sequencing (Edman degradation or mass spectrometry), provide useful alternatives. However, none of these methods are very parallel or high throughput.

[0010] Edman degradation-based peptide sequencing was first proposed by Pehr Edman in 1950; it involves the stepwise degradation of the N-terminal amino acids on a peptide through a series of chemical modifications and downstream HPLC analysis (later replaced by mass spectrometry). In the first step, the N-terminal amino acids on a peptide are degraded under mildly alkaline conditions (NMP / methanol / H 2 O) The N-terminal amino acid is modified with phenyl isothiocyanate (PITC) to form a phenylthiocarbamoyl (PTC) derivative. In the second step, the PTC-modified amino group is treated with acid (anhydrous TFA) to produce a cleaved cyclic ATZ (2-anilino-5(4)-thiooxazolinone) modified amino acid, leaving a new N-terminus on the peptide. The cleaved cyclic ATZ-amino acid is converted to a PTH-amino acid derivative and analyzed by reverse phase HPLC. The process continues in an iterative manner until all or part of the number of amino acids comprising the peptide sequence are removed from the N-terminus and identified. Typically, Edman degradation peptide sequencing is slow and has a limited throughput of only a small number of peptides per day.

[0011] In the past 10-15 years, peptide analysis using MALDI, electrospray mass spectrometry (MS), and LC-MS / MS has largely replaced Edman degradation. Despite recent advances in MS instrumentation (Riley et al., 2016, Cell Syst 2:142-143), MS still has some disadvantages, including high instrument cost, high technical requirements for users, poor quantitative capabilities, and limited measurement capabilities across the dynamic range of the proteome. For example, absolute quantification and even relative quantification between samples are challenging due to different levels of protein ionization efficiency. The use of mass tags can help improve relative quantification, but requires labeling of the proteome. Dynamic range is another difficulty, where the concentration of proteins in a sample can vary over a wide range (over 10 orders of magnitude for plasma). MS typically analyzes only high-abundance species, making the characterization of low-abundance proteins very challenging. Finally, sample throughput is usually limited to a few thousand peptides per run, which is insufficient for true bottom-up high-throughput proteome analysis for data-independent analysis (DIA). Furthermore, there are significant computational requirements for the deconvolution of the thousands of complex MS spectra recorded for each sample.

[0012] Therefore, there is still a need in the art for improved techniques related to macromolecular sequencing and / or analysis, applied to protein sequencing and / or analysis, and products, methods and kits for implementing the same. There is a need for highly parallelized, accurate, sensitive and high-throughput proteomics techniques. The present invention provides methods and apparatus having these advantages and other benefits.

[0013] Various aspects of the invention will become apparent by reference to the following detailed description.To this end, various references are set forth herein that describe in more detail certain background information, processes, compounds and / or compositions, and each is incorporated herein by reference in its entirety. Summary of the invention

[0014] Embodiments of the present disclosure generally relate to methods for highly parallel, high-throughput digital macromolecular analysis, particularly peptide analysis.

[0015] In a first embodiment is a method of analyzing a macromolecule comprising the steps of:

[0016] (a) providing a macromolecule and an associated record label attached to a solid support;

[0017] (b) contacting the macromolecule with a first binding agent capable of binding to the macromolecule, wherein the first binding agent comprises a first coding tag having identification information about the first binding agent;

[0018] (c) transferring the information of the first encoding label to the recording label to generate a first sequence extended recording label;

[0019] (d) contacting the macromolecule with a second binding agent capable of binding to the macromolecule, wherein the second binding agent comprises a second coding tag having identification information of the second binding agent;

[0020] (e) transferring the information of the second encoded label to the first sequence extended recording label to generate a second sequence extended recording label; and

[0021] (f) Analyzing the second order extension record tag.

[0022] In a second embodiment is the process of the first embodiment wherein contacting steps (b) and (d) are performed sequentially.

[0023] In a third embodiment is the process of the first embodiment wherein contacting steps (b) and (d) are performed simultaneously.

[0024] In a fourth embodiment, the method of the first embodiment further includes the following steps between steps (e) and (f):

[0025] (x) repeating steps (d) and (e) one or more times by replacing the second binding agent with a third (or higher order) binding agent capable of binding to the macromolecule, wherein the third (or higher order) binding agent comprises a third (or higher order) coding tag having identification information of the third (or higher order) binding agent; and

[0026] (y) transferring information of the third (or higher order) coded label to the second (or higher order) extended recording label to generate a third (or higher order) extended recording label;

[0027] And wherein in step (f) the third (or higher order) extended record tag is analyzed.

[0028] In a fifth embodiment is a method of analyzing a macromolecule comprising the steps of:

[0029] (a) providing a macromolecule, an associated first recording tag and an associated second recording tag attached to a solid support;

[0030] (b) contacting the macromolecule with a first binding agent capable of binding to the macromolecule, wherein the first binding agent comprises a first coding tag having identification information about the first binding agent;

[0031] (c) transferring the information of the first encoding label to the first recording label to generate a first sequence extended recording label;

[0032] (d) contacting the macromolecule with a second binding agent capable of binding to the macromolecule, wherein the second binding agent comprises a second coding tag having identification information of the second binding agent;

[0033] (e) transferring the information of the second encoding tag to the second recording tag to generate a second sequence extended recording tag; and

[0034] (f) Analyzing the first and second extended record labels.

[0035] In a sixth embodiment is the method of the fifth embodiment wherein contacting steps (b) and (d) are performed in tandem.

[0036] In a seventh embodiment is the method of the fifth embodiment wherein contacting steps (b) and (d) are performed simultaneously.

[0037] In an eighth embodiment is the method of the fifth embodiment, wherein step (a) further comprises providing an associated third (or higher order) record label attached to the solid support.

[0038] In a ninth embodiment, the method of the eighth embodiment further includes the following steps between steps (e) and (f):

[0039] (x) repeating steps (d) and (e) one or more times by replacing the second binding agent with a third (or higher order) binding agent capable of binding to the macromolecule, wherein the third (or higher order) binding agent comprises a third (or higher order) coding tag having identification information of the third (or higher order) binding agent; and

[0040] (y) transferring information of the third (or higher order) encoded label to the third (or higher order) recording label to generate a third (or higher order) extended recording label;

[0041] And wherein in step (f) the first, second and third (or higher order) extended record tags are analyzed.

[0042] In the tenth embodiment is the method of any one of the fifth to ninth embodiments, wherein the first encoding tag, the second encoding tag and any higher order encoding tags comprise a bound cycle-specific spacer sequence.

[0043] In the eleventh embodiment, is a method for analyzing peptides, comprising the steps of:

[0044] (a) providing a peptide and an associated recording tag attached to a solid support;

[0045] (b) modifying the N-terminal amino acid (NTAA) of the peptide using a chemical reagent;

[0046] (c) contacting the peptide with a first binding agent capable of binding to the modified NTAA, wherein the first binding agent comprises a first coding tag having identification information of the first binding agent;

[0047] (d) transferring the information of the first encoding label to the recording label to generate an extended recording label; and

[0048] (e) Analyzing the extended record tag.

[0049] In the 12th embodiment is the method of the 11th embodiment, wherein step (c) further comprises contacting the peptide with a second (or higher order) binding agent comprising a second (or higher order) coding tag, wherein the second (or higher order) coding tag has identification information of the second (or higher order) binding agent, wherein the second (or higher order) binding agent is capable of binding to a modified NTAA other than the modified NTAA of step (b).

[0050] In a thirteenth embodiment is the method of the twelfth embodiment, wherein after contacting said peptide with said first binding agent, said peptide is subsequently contacted with a second (or higher order) binding agent.

[0051] In a fourteenth embodiment is the method of the twelfth embodiment, wherein contacting said peptide with a second (or higher order) binding agent and contacting said peptide with said first binding agent occur simultaneously.

[0052] In the 15th embodiment is the method of any one of the 11th to 14th embodiments, wherein the chemical reagent is an isothiocyanate derivative, 2,4-dinitrobenzenesulfonic acid (DNBS), 4-sulfonyl-2-nitrofluorobenzene (SNFB) 1-fluoro-2,4-dinitrobenzene, dansyl chloride, 7-methoxycoumarin acetic acid, a thioacylation agent, a thioacetylation agent or a thiobenzylation agent.

[0053] In the 16th embodiment is a method for analyzing peptides, comprising the steps of:

[0054] (a) providing a peptide and an associated recording tag attached to a solid support;

[0055] (b) modifying the N-terminal amino acid (NTAA) of the peptide with a chemical reagent to produce a modified NTAA;

[0056] (c) contacting the peptide with a first binding agent capable of binding to the modified NTAA, wherein the first binding agent comprises a first coding tag having identification information of the first binding agent;

[0057] (d) transferring information of the first encoding label to the recording label to generate a first sequence extended recording label;

[0058] (e) removing the modified NTAA to expose the new NTAA;

[0059] (f) modifying the new NTAA using a chemical reagent to produce a new modified NTAA;

[0060] (g) contacting the peptide with a second binding agent capable of binding to the modified NTAA, wherein the second binding agent comprises a second coding tag having identification information of the second binding agent;

[0061] (h) transferring the information of the second encoded label to the first sequence extended recording label to generate a second sequence extended recording label; and

[0062] (i) Analyzing the second extended record label.

[0063] In the 17th embodiment is a method for analyzing peptides, comprising the steps of:

[0064] (a) providing a peptide and an associated recording tag attached to a solid support;

[0065] (b) contacting the peptide with a first binding agent capable of binding to the N-terminal amino acid (NTAA) of the peptide, wherein the first binding agent comprises a first coding tag having identification information of the first binding agent;

[0066] (c) transferring the information of the first encoding label to the recording label to generate an extended recording label; and

[0067] (d) Analyzing the extended record tag.

[0068] In the 18th embodiment is the method of the 17th embodiment, wherein step (b) further comprises contacting the peptide with a second (or higher order) binding agent comprising a second (or higher order) coding tag having identification information about the second (or higher order) binding agent, wherein the second (or higher order) binding agent is capable of binding to an NTAA other than the NTAA of the peptide.

[0069] In a nineteenth embodiment is the method of the eighteenth embodiment, wherein after contacting said peptide with said first binding agent, said peptide is subsequently contacted with a second (or higher order) binding agent.

[0070] In the twentieth embodiment is the method of the eighteenth embodiment, wherein contacting the peptide with a second (or higher order) binding agent and contacting the peptide with the first binding agent occur simultaneously

[0071] In the 21st embodiment, is a method for analyzing a peptide, comprising the steps of:

[0072] (a) providing a peptide and an associated recording tag attached to a solid support;

[0073] (b) contacting the peptide with a first binding agent capable of binding to the N-terminal amino acid (NTAA) of the peptide, wherein the first binding agent comprises a first coding tag having identification information of the first binding agent;

[0074] (c) transferring information of the first encoding label to the recording label to generate a first sequence extended recording label;

[0075] (d) removing the NTAA of the peptide to reveal a new NTAA;

[0076] (e) contacting the peptide with a second binding agent capable of binding to the novel NTAA, wherein the second binding agent comprises a second coding tag having identification information of the second binding agent;

[0077] (h) transferring the information of the second encoded label to the first sequence extended recording label to generate a second sequence extended recording label; and

[0078] (i) Analyzing the second extended record label.

[0079] In a twenty-second embodiment, it is the method of any one of embodiments one to ten, wherein the macromolecule is a protein, polypeptide, or peptide.

[0080] In a twenty-third embodiment, it is the method of any one of embodiments 1 to 10, wherein the macromolecule is a peptide.

[0081] In the 24th embodiment, it is the method of any one of the 1st to 23rd embodiments, wherein the peptide is obtained by fragmenting a protein isolated from a biological sample.

[0082] In a twenty-fifth embodiment, is the method of any one of embodiments 1 to 10, wherein the macromolecule is a lipid, a carbohydrate, or a macrocycle.

[0083] In the 26th embodiment, it is the method of any one of the 1st to 25th embodiments, wherein the recording label is a DNA molecule, a DNA having a pseudo-complementary base, an RNA molecule, a BNA molecule, an XNA molecule, an LNA molecule, a PNA molecule, a γPNA molecule or a combination thereof.

[0084] In a 27th embodiment, it is the method of any one of the 1st to 26th embodiments, wherein the record label comprises a universal priming site.

[0085] In the 28th embodiment, is the method of the 27th embodiment, wherein the universal priming site comprises a primer site for amplification, sequencing, or both.

[0086] In a 29th embodiment, it is the method of any one of embodiments 1 to 28, wherein the record tag comprises a unique molecular identifier (UMI).

[0087] In a 30th embodiment, it is the method of any one of embodiments 1 to 29, wherein the record label comprises a bar code.

[0088] In a 31st embodiment, it is the method of any one of the 1st to 30th embodiments, wherein the record tag comprises a spacer at its 3'-end.

[0089] In a 32nd embodiment, it is the method of any one of embodiments 1 to 31, wherein said macromolecule and said associated recording tag are covalently attached to said solid support.

[0090] In the 33rd embodiment, it is the method of any one of the 1st to 32nd embodiments, wherein the solid support is a bead, a porous bead, a porous matrix, an array, a glass surface, a silicon surface, a plastic surface, a filter, a membrane, nylon, a silicon crystal chip, a flow chip, a biochip including a signal transduction electronic device, a microtiter well, an ELISA plate, a rotating interferometry disk, a nitrocellulose membrane, a nitrocellulose-based polymer surface, a nanoparticle or a microsphere.

[0091] In the 34th embodiment, it is the method of the 33rd embodiment, wherein the solid support is polystyrene beads, polymer beads, agarose beads, acrylamide beads, solid core beads, porous beads, paramagnetic beads, glass beads, or controlled pore beads.

[0092] In a 35th embodiment, it is the method of any one of embodiments 1 to 34, wherein a plurality of macromolecules and associated record tags are bound to the solid support.

[0093] In a 36th embodiment, is the method of the 35th embodiment, wherein the plurality of macromolecules are spaced apart on the solid support at an average distance of >50 nm.

[0094] In a 37th embodiment, it is the method of any one of embodiments 1 to 36, wherein the binding agent is a polypeptide or protein.

[0095] In the 38th embodiment, is the method of the 37th embodiment, wherein the binding agent is a modified aminopeptidase, a modified aminoacyl-tRNA synthetase, a modified anticalin, or a modified ClpS.

[0096] In a 39th embodiment, it is the method of any one of embodiments 1 to 38, wherein said binding agent is capable of selectively binding said macromolecule.

[0097] In a 40th embodiment, it is the method of any one of the 1st to 39th embodiments, wherein the record tag is a DNA molecule, an RNA molecule, a BNA molecule, an XNA molecule, an LNA molecule, a PNA molecule, a γPNA molecule or a combination thereof.

[0098] In a 41st embodiment, it is the method of any one of embodiments 1 to 40, wherein the record tag comprises a coding sequence.

[0099] In a 42nd embodiment, it is the method of any one of embodiments 1 to 41, wherein the record tag further comprises a spacer, a binding cycle specific sequence, a unique molecular identifier, a universal priming site, or any combination thereof.

[0100] In a 43rd embodiment, it is the method of any one of embodiments 1 to 42, wherein the binding agent and the coding tag are connected by a linker.

[0101] In the 44th embodiment, it is the method of any one of embodiments 1 to 42, wherein the binding agent and the encoding tag are linked by a SpyTag / SpyCatcher or SnoopTag / SnoopCatcher peptide-protein pair.

[0102] In a 45th embodiment, it is the method of any one of the 1st to 44th embodiments, wherein the transfer of the information of the encoding tag to the recording tag is mediated by DNA ligase.

[0103] In a 46th embodiment, it is the method of any one of the 1st to 44th embodiments, wherein the transfer of the information of the encoding tag to the recording tag is mediated by a DNA synthesizer.

[0104] In a 47th embodiment, it is the method of any one of embodiments 1 to 44, wherein the transfer of the information of the encoding tag to the recording tag is mediated by chemical linkage.

[0105] In a 48th embodiment, it is the method of any one of embodiments 1 to 47, wherein analyzing the extended record tag comprises a nucleic acid sequencing method. In a 49th embodiment, it is the method of embodiment 48, wherein the nucleic acid sequencing method is by synthesis sequencing, ligation sequencing, hybridization sequencing, polony sequencing, ion semiconductor sequencing or pyrophosphate sequencing.

[0106] In a 50th embodiment, it is the method according to the 48th embodiment, wherein the nucleic acid sequencing method is single molecule real-time sequencing, nanopore-based sequencing, or direct imaging of DNA using advanced microscopy.

[0107] In a 51st embodiment, it is the method of any one of embodiments 1 to 50, wherein the extended record tag is amplified prior to analysis.

[0108] In a 52nd embodiment, it is the method of any one of the 1st to 51st embodiments, wherein the order of the encoded tag information contained in the extended record labels provides information about the binding order of the binding agents bound to the macromolecule.

[0109] In a 53rd embodiment, it is the method of any one of the 1st to 52nd embodiments, wherein the frequency of the encoded tag information contained in the extended recording label provides information about the binding frequency of the binding agent bound to the macromolecule.

[0110] In a 54th embodiment, it is the method of any one of the 1st to 53rd embodiments, wherein a plurality of extended record tags representing a plurality of macromolecules are analyzed in parallel.

[0111] In a 55th embodiment, is the method of the 54th embodiment, wherein a plurality of extended record labels representing a plurality of macromolecules are analyzed in a multiplexed assay.

[0112] In a 56th embodiment, it is the method of any one of embodiments 1 to 55, wherein the plurality of extended record tags are subjected to target abundance detection prior to analysis.

[0113] In a 57th embodiment, it is the method of any one of the 1st to 56th embodiments, wherein the plurality of extended record tags are subjected to subtractive analysis prior to analysis.

[0114] In a 58th embodiment, it is the method of any one of the 1st to 57th embodiments, wherein the plurality of extended recording tags are normalized prior to analysis to reduce high abundance species.

[0115] In the 59th embodiment, it is the method of any one of embodiments 1 to 58, wherein the NTAA is removed using a modified aminopeptidase, a modified amino acid tRNA synthetase, mild Edman degradation, Edmanase, or anhydrous TFA.

[0116] In the 60th embodiment, it is the method of any one of embodiments 1 to 59, wherein the at least one binding agent binds to the terminal amino acid residue.

[0117] In a 61st embodiment, it is the method of any one of embodiments 1 to 60, wherein the at least one binding agent binds to a post-translationally modified amino acid.

[0118] In a 62nd embodiment, one or more peptides from a sample comprising a plurality of protein complexes, proteins, or polypeptides are analyzed, the method comprising:

[0119] (a) partitioning a plurality of protein complexes, proteins or polypeptides within a sample into a plurality of compartments, wherein each compartment comprises a plurality of compartment tags, which are optionally attached to a solid support, wherein the plurality of compartment tags within a single compartment are identical and different from the compartment tags of other compartments;

[0120] (b) fragmenting a plurality of protein complexes, proteins and / or polypeptides into a plurality of peptides;

[0121] (c) contacting the plurality of peptides with the plurality of compartment tags under conditions sufficient to allow the plurality of peptides to anneal or link to the plurality of compartment tags within the plurality of compartments, thereby producing a plurality of compartment-tagged peptides;

[0122] (d) collecting compartment-labeled peptides from the plurality of compartments; and

[0123] (e) analyzing one or more compartment labeled peptides according to the method of any one of embodiments 1 to 21 and embodiments 26 to 61.

[0124] In a 63rd embodiment, the method of the 62nd embodiment, the compartment is a microfluidic droplet.

[0125] In a 64th embodiment, is the method of the 62nd embodiment, wherein the compartment is a microwell.

[0126] In a 65th embodiment, is the method according to the 62nd embodiment, wherein the compartments are separate areas on the surface.

[0127] In a 66th embodiment, it is the method of any one of embodiments 62 to 65, wherein each compartment contains an average of one single cell.

[0128] In a 67th embodiment, one or more peptides from a sample comprising a plurality of protein complexes, proteins, or polypeptides are analyzed, the method comprising:

[0129] (a) Labeling multiple protein complexes, proteins or peptides with multiple universal DNA tags;

[0130] (b) partitioning a plurality of protein complexes, proteins or polypeptides within the sample into a plurality of compartments, wherein each compartment comprises a plurality of compartment tags, wherein the plurality of compartment tags within a single compartment are the same and different from the compartment tags of other compartments;

[0131] (c) contacting the plurality of protein complexes, proteins, or polypeptides with the plurality of compartment tags under conditions sufficient to allow the plurality of peptides to anneal or link to the plurality of compartment tags within the plurality of compartments, thereby producing a plurality of compartment-tagged protein complexes, proteins, or polypeptides;

[0132] (d) collecting compartment-labeled protein complexes, proteins, or polypeptides from the plurality of compartments;

[0133] (e) optionally fragmenting the compartment-tagged protein complex, protein or polypeptide into compartment-tagged peptides; and

[0134] (f) analyzing one or more compartment labeled peptides according to the method of any one of embodiments 1 to 21 and embodiments 26 to 61.

[0135] In a 68th embodiment, it is the method of any one of embodiments 62 to 67, wherein the compartment label information is transferred to a record tag associated with the peptide by primer extension or ligation.

[0136] In a 69th embodiment, it is the method of any one of embodiments 62 to 68, wherein the solid support comprises beads.

[0137] In the 70th embodiment, it is the method of the 69th embodiment, wherein the beads are polystyrene beads, polymer beads, agarose beads, acrylamide beads, solid core beads, porous beads, paramagnetic beads, glass beads or controlled pore beads.

[0138] In the 71st embodiment, it is the method of any one of embodiments 62 to 70, wherein the compartment tag comprises a single-stranded or double-stranded nucleotide molecule.

[0139] In a 72nd embodiment, it is the method of any one of embodiments 62 to 71, wherein the compartment label comprises a barcode and optionally a UMI.

[0140] In the 73rd embodiment, it is the method of the 72nd embodiment, wherein the solid support is a bead, the compartment tag comprises a barcode, and further wherein the bead comprising a plurality of compartment tags attached thereto is formed by mix-and-split synthesis.

[0141] In the 74th embodiment, it is the method of the 72nd embodiment, wherein the solid support is a bead, the compartment tag comprises a barcode, and further wherein the bead comprising a plurality of compartment tags attached thereto is formed by individual synthesis or immobilization.

[0142] In the 75th embodiment, it is the method of any one of the 62nd to 74th embodiments, wherein the spacer tag is a component in a record label, and the record label optionally further includes a spacer, a specific molecular tag, a universal priming site, or any combination thereof.

[0143] In the 76th embodiment, it is the method of any one of the 62nd to 75th embodiments, wherein the spacer tag further comprises a functional group capable of reacting with an internal amino acid or an N-terminal amino acid on the plurality of protein complexes, proteins, or polypeptides.

[0144] In the 77th embodiment, which is the method of the 76th embodiment, the functional group is an NHS group.

[0145] In the 78th embodiment, it is the method of the 76th embodiment, wherein the functional group is an aldehyde group.

[0146] The 79th embodiment is the method of any one of the 62nd to 78th embodiments, wherein the plurality of compartment labels are formed by the following method:

[0147] The compartment labels are printed, dotted, inkjetted into the compartments, or a combination thereof.

[0148] In the 80th embodiment, it is the method of any one of embodiments 62 to 79, wherein the compartment tag further comprises a peptide.

[0149] In the 81st embodiment, it is the method of the 80th embodiment, wherein the compartment tag peptide comprises a protein ligase recognition sequence.

[0150] The 82nd embodiment is the method of the 81st embodiment, wherein the protein ligase is butelase I or a homolog thereof.

[0151] In the 83rd embodiment, it is the method of any one of embodiments 62nd to 82nd, wherein the plurality of polypeptides are fragmented with a protease.

[0152] In the 84th embodiment, is the method of the 83rd embodiment, wherein the protease is a metalloprotease.

[0153] In the 85th embodiment, is the method of the 84th embodiment, wherein the activity of the metalloprotease is regulated by photon-stimulated release of metal cations.

[0154] In the 86th embodiment, it is the method of any one of the 62nd to 85th embodiments, further comprising subtracting one or more high abundance proteins from the sample before partitioning the plurality of polypeptides into the plurality of compartments.

[0155] In the 87th embodiment, it is the method of any one of embodiments 62 to 86, further comprising releasing the compartment tag from the solid support before associating the plurality of peptides to the compartment tag.

[0156] In the 88th embodiment, it is the method of the 62nd embodiment, further comprising the step (d) of linking the compartment tag peptide to a solid support associated with the record tag.

[0157] In the 89th embodiment, it is the method of the 88th embodiment, further comprising transmitting the information of the compartment label on the compartment-tagged peptide to an associated recording label.

[0158] In the 90th embodiment, it is the method of the 89th embodiment, further comprising removing the compartment tag from the compartment-tagged peptide before step (e).

[0159] In the 91st embodiment, it is the method of any one of the 62nd to 90th embodiments, further comprising determining the identity of the single cell from which the analyzed peptide is derived based on the compartment tag sequence of the analyzed peptide.

[0160] In the 92nd embodiment, it is a method of any one of the 62nd to 90th embodiments, further comprising determining the identity of the protein or protein complex from which the analyzed peptide is derived based on the compartment tag sequence of the analyzed peptide.

[0161] In the 93rd embodiment is a method for analyzing a plurality of macromolecules comprising the steps of:

[0162] (a) providing a plurality of macromolecules and associated record labels attached to a solid support;

[0163] (b) contacting the plurality of macromolecules with a plurality of binding agents capable of binding to the plurality of macromolecules, wherein each binding agent comprises a coding tag having identification information of the binding agent;

[0164] (c) (i) transferring the information of the macromolecule-associated recording tag to the coding tag of the binding agent bound to the macromolecule to produce an extended coding tag; or (ii) transferring the information of the macromolecule-associated recording tag and the coding tag of the binding agent bound to the macromolecule to a di-tag construct;

[0165] (d) collecting the extended coding tag or di-tag construct;

[0166] (e) optionally repeating steps (b)-(d) for one or more binding cycles;

[0167] (f) analyzing the pool of extended encoding tags or di-tag constructs.

[0168] In a 94th embodiment, is the method of the 93rd embodiment, wherein the macromolecule is a protein.

[0169] In a 95th embodiment, is the method of the 93rd embodiment, wherein the macromolecule is a peptide.

[0170] In a 96th embodiment, is the method according to the 95th embodiment, wherein the peptide is obtained by fragmenting a protein isolated from a biological sample.

[0171] In the 97th embodiment, it is the method of any one of the 93rd to 96th embodiments, wherein the record label is a DNA molecule, an RNA molecule, a PNA molecule, a BNA molecule, an XNA molecule, an LNA molecule, a γPNA molecule or a combination thereof.

[0172] In a 98th embodiment, it is the method of any one of 93rd to 97th implementations, wherein the record tag comprises a unique molecular identifier (UMI).

[0173] In a 99th embodiment, it is the method of any one of embodiments 93-98, wherein the record label comprises a compartment label.

[0174] In the 100th embodiment, it is the method of any one of the 93rd to 99th embodiments, wherein the record label comprises a universal priming site.

[0175] In the 101st embodiment is the method of any one of embodiments 93-100, wherein the record tag comprises a spacer at its 3'-end.

[0176] In the 102nd embodiment, it is a method according to any one of embodiments 93 to 101, wherein the 3'-end of the record tag is closed to prevent extension of the record tag by a synthetase, and information of the record tag associated with the macromolecule and information of the encoding tag of the binding agent bound to the macromolecule are transmitted to the di-tag construct.

[0177] In the 103rd embodiment, it is the method of any one of embodiments 93rd to 102nd, wherein the coding tag comprises a coding sequence.

[0178] In a 104th embodiment, it is the method of any one of embodiments 93 to 103, wherein the encoded tag comprises a UMI.

[0179] In the 105th embodiment, it is the method of any one of embodiments 93rd to 104th, wherein the encoding tag comprises a universal priming site.

[0180] In the 106th embodiment, the method according to any one of embodiments 93-105, wherein the coding tag comprises a spacer at its 3'-end.

[0181] In the 107th embodiment, it is the method of any one of embodiments 93 to 106, wherein the encoding tag comprises a binding cycle-specific sequence.

[0182] In the 108th embodiment, it is the method of any one of embodiments 93 to 107, wherein the binding agent and the coding tag are connected by a linker.

[0183] In a 109th embodiment, it is the method of any one of the 93rd to 108th embodiments, wherein the transfer of the information of the recording tag to the encoding tag is achieved by primer extension.

[0184] In the 110th embodiment, it is the method of any one of the 93rd to 108th embodiments, wherein the transmitting of the information of the recording tag to the encoding tag is achieved by connection.

[0185] In the 111th embodiment, it is the method of any one of the 93rd to 108th embodiments, wherein the di-tag construct is generated by gap filling, primer extension, or both.

[0186] In the 112th embodiment is a method of any one of embodiments 93-97, 107, 108 and 111, wherein the di-tag molecule comprises a universal priming site derived from the recording tag, a compartment tag derived from the recording tag, a unique molecular identifier derived from the recording tag, an optional spacer derived from the recording tag, a coding sequence derived from the coding tag, a unique molecular identifier derived from the coding tag, an optional spacer derived from the coding tag and a universal priming site derived from the coding tag.

[0187] In a 113th embodiment, it is the method of any one of embodiments 93-112, wherein the macromolecule and associated record tag are covalently bound to the solid support.

[0188] In the 114th embodiment, it is the method of the 113th embodiment, wherein the solid support is a bead, a porous bead, a porous matrix, an array, a glass surface, a silicon surface, a plastic surface, a filter, a membrane, nylon, a silicon crystal chip, a flow chip, a biochip containing a signal transduction electronic device, a microtiter well, an ELISA plate, a rotating interferometry disk, a nitrocellulose membrane, a nitrocellulose-based polymer surface, a nanoparticle or a microsphere.

[0189] The 115th embodiment is a method according to the 114th embodiment, wherein the solid support is polystyrene beads, polymer beads, agarose beads, acrylamide beads, solid core beads, porous beads, paramagnetic beads, glass beads, or controlled pore beads.

[0190] The 116th embodiment is a method according to any one of embodiments 93 to 115, wherein the binding agent is a polypeptide or protein.

[0191] The 117th embodiment is a method according to the 116th embodiment, wherein the binding agent is a modified aminopeptidase, a modified aminoacyl-tRNA synthetase, a modified anticalin, or an antibody or a binding fragment of an antibody.

[0192] The 118th embodiment is the method of any one of embodiments 95 to 117, wherein the binding agent binds to a single amino acid residue, a dipeptide, a tripeptide, or a post-translational modification of a peptide.

[0193] The 119th embodiment is a method according to the 118th embodiment, wherein the binding agent is bound to the N-terminal amino acid residue, the C-terminal amino acid residue or an internal amino acid residue.

[0194] The 120th embodiment is a method according to the 118th embodiment, wherein the binding agent binds to the N-terminal peptide, the C-terminal peptide, or an internal peptide.

[0195] The 121st embodiment is a method according to the 119th embodiment, wherein the binding agent binds to the N-terminal amino acid residue and cleaves the N-terminal amino acid residue after each binding cycle.

[0196] The 122nd embodiment is a method according to the 119th embodiment, wherein the binding agent binds to the C-terminal amino acid residue and cleaves the C-terminal amino acid residue after each binding cycle.

[0197] Embodiment 123 is the method of embodiment 121, wherein the N-terminal amino acid residue is cleaved by Edman degradation.

[0198] Embodiment 124 is the method of embodiment 93, wherein the binding agent is a site-specific covalent label of an amino acid or a post-translational modification.

[0199] Embodiment 125 is the method of any one of embodiments 93 to 124, wherein after step (b), the complex comprising the macromolecule and the associated binding agent is dissociated from the solid support and distributed into droplets or an emulsion of microfluidic droplets.

[0200] Embodiment 126 is the method of embodiment 125, wherein each of the microfluidic droplets, on average, contains a complex comprising a macromolecule and a binding agent.

[0201] Embodiment 127 is the method of embodiment 125 or 126, wherein the record tag is amplified prior to generating the extended encoding tag or di-tag construct.

[0202] Embodiment 128 is the method of any one of embodiments 125 to 127, wherein the emulsion fusion PCR is used to transfer the record tag information to the encoding tag or to create a set of di-tag constructs.

[0203] Embodiment 129 is the method of any one of embodiments 93 to 128, wherein the collection of extended encoding tags or di-tag constructs is amplified prior to analysis.

[0204] Embodiment 130 is the method of any one of embodiments 93 to 129, wherein said analyzing the collection of extended encoding tags or di-tag constructs comprises a nucleic acid sequencing method.

[0205] Embodiment 131 is the method described in embodiment 130, wherein the nucleic acid sequencing method is by synthesis sequencing, ligation sequencing, hybridization sequencing, polony sequencing, ion semiconductor sequencing or pyrophosphate sequencing.

[0206] Embodiment 132 is the method described in embodiment 130, wherein the nucleic acid sequencing method is single molecule real-time sequencing, nanopore-based sequencing, or direct imaging of DNA using advanced microscopy.

[0207] Embodiment 133 is the method of embodiment 130, wherein the partial composition of the macromolecule is determined by analyzing multiple extended encoding tags or di-tag constructs using unique compartment labels and optional UMIs.

[0208] Embodiment 134 is the method of any one of embodiments 1 to 133, wherein the analyzing step is performed using a sequencing method having a per-base error rate of >5%, >10%, >15%, >20%, >25% or >30%.

[0209] Embodiment 135 is the method of any one of embodiments 1 to 134, wherein the identification component of the encoding label, the recording label, or both comprises an error correction code.

[0210] Embodiment 136 is the method of embodiment 135, wherein the identification component is selected from an encoder sequence, a barcode, a UMI, a compartment tag, a cycle-specific sequence, or any combination thereof.

[0211] Embodiment 137 is the method described in embodiment 135 or 136, wherein the error correction code is selected from Hamming code, Lie distance code, asymmetric Lie distance code, Reed-Solomon code and Levenshtein-Tenengolts code.

[0212] Embodiment 138 is a method of any one of embodiments 1 to 134, wherein the identification component of the coding tag, the recording tag, or both is capable of generating a unique current or ion flux or optical signature, and wherein the analysis step includes detecting the unique current or ion flux or optical signature to identify the identification component.

[0213] Embodiment 139 is the method of embodiment 138, wherein the identification component is selected from an encoder sequence, a barcode, a UMI, a compartment tag, a cycle-specific sequence, or any combination thereof.

[0214] Embodiment 140 is a method for analyzing a plurality of macromolecules, comprising the steps of:

[0215] (a) providing a plurality of macromolecules and associated record labels attached to a solid support;

[0216] (b) contacting the plurality of macromolecules with a plurality of binding agents capable of binding to homologous macromolecules, wherein each binding agent comprises a coded tag having identification information of the binding agent;

[0217] (c) transferring information of a first coding tag of a first binding agent to a first recording tag associated with a first macromolecule to generate a first sequence-extended recording tag, wherein the first binding agent is bound to the first macromolecule;

[0218] (d) contacting the plurality of macromolecules with a plurality of binding agents capable of binding to homologous macromolecules;

[0219] (e) transferring information of a second coding tag of a second binding agent to the first sequence-extended recording tag to generate a second sequence-extended recording tag, wherein the second binding agent binds to the first macromolecule;

[0220] (f) optionally repeating steps (d)-(e) for "n" binding cycles, wherein the information of each encoded tag of each binding agent bound to the first macromolecule is transferred to the extended record tag generated in the previous binding cycle to generate an nth order extended record tag representing the first macromolecule;

[0221] (g) Analyze the n-th order extended record tag.

[0222] Embodiment 141 is the method described in embodiment 140, wherein a plurality of n-th order extended record labels representing a plurality of macromolecules are generated and analyzed.

[0223] Embodiment 142 is the method of embodiment 140 or 141, wherein the macromolecule is a protein.

[0224] Embodiment 143 is the method of embodiment 142, wherein the macromolecule is a peptide.

[0225] Embodiment 144 is the method of embodiment 143, wherein the peptide is obtained by fragmenting and separating proteins of a biological sample.

[0226] Embodiment 145 is the method of any one of embodiments 140 to 144, wherein the plurality of macromolecules comprises macromolecules from a plurality of pooled samples.

[0227] Embodiment 146 is the method of any one of embodiments 140 to 145, wherein the record label is a DNA molecule, an RNA molecule, a PNA molecule, a BNA molecule, an XNA molecule, an LNA molecule, a γPNA molecule, or a combination thereof.

[0228] Embodiment 147 is the method of any one of embodiments 140 to 146, wherein the record tag comprises a unique molecular identifier (UMI).

[0229] Embodiment 148 is the method of embodiments 140 to 147, wherein the record label comprises a compartment label.

[0230] Embodiment 149 is the method of any one of embodiments 140 to 148, wherein the record label comprises a universal priming site.

[0231] Embodiment 150 is the method of any one of embodiments 140 to 149, wherein the record tag comprises a spacer at its 3'-end.

[0232] Embodiment 151 is the method of any one of embodiments 140 to 150, wherein the encoding tag comprises an encoder sequence.

[0233] Implementation Example 152 is the method of any one of Implementation Examples 140 to 151, wherein the encoded tag comprises a UMI.

[0234] Embodiment 153 is the method of any one of embodiments 140 to 152, wherein the encoding tag comprises a universal priming site.

[0235] Embodiment 154 is the method of any one of embodiments 140 to 153, wherein the encoding tag comprises a spacer at its 3 '-end.

[0236] Embodiment 155 is the method of any one of embodiments 140 to 154, wherein the encoding tag comprises a binding cycle-specific sequence.

[0237] Embodiment 156 is the method of any one of embodiments 140 to 155, wherein the encoding tag comprises a specific molecular tag.

[0238] Embodiment 157 is the method of any one of embodiments 140 to 156, wherein the binding agent and the coding tag are connected via a linker.

[0239] Embodiment 158 ​​is the method of any one of embodiments 140 to 157, wherein the information transfer from the recording tag to the encoding tag is mediated by primer extension.

[0240] Embodiment 159 The method of any one of Embodiments 140 to 158, wherein the information transfer from the recording tag to the encoding tag is mediated by ligation.

[0241] Embodiment 160 is the method of any one of embodiments 140 to 159, wherein the plurality of macromolecules and associated record tags or both are bound to the solid support.

[0242] Embodiment 161 is a method according to any one of embodiments 140 to 160, wherein the solid support is a bead, a porous bead, a porous matrix, an array, a glass surface, a silicon surface, a plastic surface, a filter, a membrane, nylon, a silicon crystal chip, a flow chip, a biochip including a signal transduction electronic device, a microtiter well, an ELISA plate, a rotating interferometry disk, a nitrocellulose membrane, a nitrocellulose-based polymer surface, a nanoparticle or a microsphere.

[0243] Embodiment 162 is the method of embodiment 161, wherein the solid support is polystyrene beads, polymer beads, agarose beads, acrylamide beads, solid core beads, porous beads, paramagnetic beads, glass beads or controlled pore beads.

[0244] Embodiment 163 is the method of any one of embodiments 140 to 162, wherein the binding agent is a polypeptide or protein.

[0245] Embodiment 164 is the method of embodiment 163, wherein the binding agent is a modified aminopeptidase, a modified aminoacyl-tRNA synthetase, a modified anticalin, or an antibody or a binding fragment of an antibody.

[0246] Embodiment 165 is the method of any one of embodiments 142 to 164, wherein the binding agent binds to a single amino acid residue, a dipeptide, a tripeptide, or a post-translational modification of a peptide.

[0247] Embodiment 166 is the method of embodiment 165, wherein the binding agent binds to the N-terminal amino acid residue, the C-terminal amino acid residue, or an internal amino acid residue.

[0248] Embodiment 167 is the method of embodiment 165, wherein the binding agent binds to an N-terminal peptide, a C-terminal peptide, or an internal peptide.

[0249] Embodiment 168 is the method of any one of embodiments 142 to 164, wherein the binding agent binds to a chemical tag of a modified N-terminal amino acid residue, a modified C-terminal amino acid residue, or a modified internal amino acid residue.

[0250] Embodiment 169 is the method of embodiment 166 or 168, wherein the binding agent is bound to the chemical tag of the N-terminal amino acid residue or the modified N-terminal amino acid residue, and the N-terminal amino acid residue is cleaved after each binding cycle.

[0251] Embodiment 170 is the method of embodiment 166 or 168, wherein the binding agent is bound to the chemical tag of the C-terminal amino acid residue or the modified C-terminal amino acid residue, and the C-terminal amino acid residue is cleaved after each binding cycle.

[0252] Embodiment 171 is the method of embodiment 169, wherein the N-terminal amino acid residue is cleaved by Edman degradation, Edman enzyme, modified aminopeptidase, or modified acyl peptide hydrolase.

[0253] Embodiment 172 is the method of embodiment 163, wherein the binding agent is a specific covalent label of an amino acid site or a site-specific covalent label of a post-translational modification.

[0254] Embodiment 173 is the method of any one of embodiments 140-172, wherein the plurality of nth order extended record tags are amplified prior to analysis.

[0255] Embodiment 174 is the method of any one of embodiments 140 to 173, wherein analyzing the n-th order extended record tag comprises a nucleic acid sequencing method.

[0256] Embodiment 175 is the method of embodiment 174, wherein a plurality of n-th order extended record labels representing a plurality of macromolecules are analyzed in parallel.

[0257] Embodiment 176 is the method described in embodiment 174 or 175, wherein the nucleic acid sequencing method is by synthesis sequencing, ligation sequencing, hybridization sequencing, polony sequencing, ion semiconductor sequencing or pyrophosphate sequencing.

[0258] Embodiment 177 is a method according to embodiment 174 or 175, wherein the nucleic acid sequencing method is single molecule real-time sequencing, nanopore-based sequencing, or direct imaging of DNA using advanced microscopy. BRIEF DESCRIPTION OF THE DRAWINGS

[0259] Non-limiting embodiments of the present invention are described by way of example with reference to the accompanying drawings, which are schematic and are not intended to be drawn to scale. For illustrative purposes, not every component is marked in each figure, nor are every component not necessary for a person of ordinary skill in the art to understand the present invention shown in each embodiment of the present invention.

[0260] Attached Figure 1A -B: Attach Figure 1A An illustration of the functional elements shown in the figures is shown.

[0261] Figure 1B A general overview of transducing protein coding into DNA coding is shown, wherein multiple proteins or polypeptides are fragmented into multiple peptides, which are then converted into a library of extended record tags to represent multiple peptides. The extended record tags constitute a library of DNA encodings representing the peptide sequences. The library can be appropriately modified to be sequenced on any next generation sequencing (NGS) platform.

[0262] Attached Figures 2A-2DAn example of protein macromolecule analysis according to the method of the present disclosure is shown, which uses a binding agent (e.g., antibodies, anticalins, N-recognins proteins (e.g., ATP-dependent Clp protease adaptor protein (ClpS)), aptamers, etc. and variants / homologs thereof) containing a coding tag, the coding tag interacts with an immobilized protein, and the immobilized protein is co-localized or co-labeled with a single or multiple recording tags. The recording tag is composed of a universal priming site, a barcode (e.g., a partition barcode, a compartment barcode, a graded barcode), an optional unique molecular identifier (UMI) sequence, and a spacer sequence (Sp) for information transfer of the coding tag. The spacer tag (Sp) can be constant between all binding cycles, binding agent specific, or binding cycle number specific. The coding tag is composed of a coding sequence that provides identification information for the binding agent, an optional UMI, and a spacer sequence that hybridizes with a complementary spacer sequence on the recording tag, so as to facilitate the transfer of the coding tag information to the recording tag (e.g., primer extension, also referred to as synthetase extension herein). Figure 2A The process of generating an extended record tag by the cognate binder and protein cyclic binding is shown, and the corresponding information is transferred from the encoding tag of the binder to the protein record tag. After a series of sequential binding and encoding tag information transfer steps, a final extended record tag is generated, which contains the binder encoding tag information, and the binder encoding tag information includes an encoder sequence from the "n" binding cycle that provides identification information for the binder (e.g., antibody 2 (Ab2), antibody 3 (Ab3), ... antibody "n" (Abn)), a barcode / optional UMI sequence from the record tag, an optional UMI sequence from the binder encoding tag, and a flanking universal primer sequence at each end of the library construct to facilitate amplification and analysis by digital next-generation sequencing. Figure 2B A schematic example of labeling a protein with a DNA barcode record tag is shown. In the upper figure, N-hydroxysuccinimide (NHS) is an amine-reactive coupling agent, and dibenzocyclooctyl (DBCO) is a tensioned alkyne for "click" coupling to the surface of a solid substrate. In this scheme, the record tag is coupled to the ε amine of the lysine (K) residue (and optional N-terminal amino acid) of the protein through the NHS group. In the bottom figure, the ε amine of the lysine (K) residue is labeled with a heterobifunctional linker NHS-alkyne to produce an alkyne "click" group. Then the azide-labeled DNA record tag can be easily connected to these reactive alkyne groups by standard click chemistry. In addition, the DNA record tag can also be designed with an orthogonal methyl tetrazine (mTet) group for downstream coupling to a TCO-derived sequencing substrate by a reverse iEDDA reaction. Figure 2CExamples of protein analysis methods using record tags are shown. In the top figure, the protein macromolecule is fixed to a solid support via a capture agent and optionally by cross-linking. The protein or the capture agent can be labeled with a record tag. In the bottom figure, the protein associated with the record tag is directly fixed to the solid support. Figure 2D An overall workflow example of a simple protein immunoassay using DNA encoding of homologous binders and sequencing of the generated extended record tags is shown. The proteins can be barcoded (i.e., indexed) by the record tags and pooled prior to cyclic binding analysis, greatly increasing sample throughput and saving binding reagents. This method is actually a digital, simpler and more scalable method for performing reverse phase protein assays (RPPA).

[0263] Figure 3 AD is a process for degradation-based peptide sequencing analysis by constructing DNA-extended record tags representing the peptide sequence. This is achieved by using a cyclic process of N-terminal amino acid (NTAA) binding on a solid support, transfer of the encoded tag information to the record tag attached to the peptide, NTAA cleavage, and Edman degradation-like method that repeats the process in a cyclic manner. An overview of exemplary construction of extended record tags from N-terminal degradation of peptides is provided: (A) the N-terminal amino acid of the peptide is labeled (e.g., with a phenylthiocarbamoyl (PTC), dinitrophenyl (DNP), sulfonyl nitrophenyl (SNP), acetyl or guanidinyl group); (B) a binding agent is shown with a coding tag associated with the NTAA bound to the label; (C) a peptide is shown bound to a solid support (e.g., a bead) and associated with a record tag (e.g., via a trifunctional linker), wherein when the binding agent is bound to the NTAA of the peptide, the information of the coding tag is transferred to the record tag (e.g., via primer extension) to produce an extended record tag; (D) the labeled NTAA is cleaved by chemical or enzymatic methods to expose a new NTAA. As indicated by the arrow, the cycle is repeated "n" times to produce the final extended record tag. The final extended record tag is optionally flanked by a universal priming site to facilitate downstream amplification and DNA sequencing. The forward universal priming site (e.g., Illumina's P5-S1 sequence) can be part of the original record tag design, and the reverse universal priming site (e.g., Illumina's P7-S2' sequence) can be added in the last step of the extension of the record tag. The last step can be done independently with a binding reagent.

[0264] Figure 4A -B shows an exemplary protein sequencing process according to the methods disclosed herein. Figure 4AAn exemplary workflow with alternative modes outlined in light grey dashed lines is shown, with specific embodiments shown as boxes linked by arrows. Alternative models for each step of the process are shown in the box under the arrow. Figure 4B The option of performing cyclic binding and coded tag information transfer steps to improve information transfer efficiency is shown. Multiple record tags can be used per molecule. In addition, for a given binding event, the coded tag information transfer to the record tag can be performed multiple times, or alternatively, a surface amplification step is used to generate copies of the extended record tag library, and so on.

[0265] Figure 5 AB shows an overview of an exemplary construction of an extended record tag using primer extension to transfer the identification information of a coding tag of a binder to a record tag associated with a macromolecule (e.g., a peptide) to produce an extended record tag. The coding tag containing a unique coding sequence with identification information about the binder is optionally flanked at each end by a common spacer sequence (Sp'). Figure 5 A shows an NTAA binder comprising a coding tag of NTAA bound to a peptide labeled with a record tag connected to a bead. The record tag is annealed with the coding tag through a complementary spacer sequence (Sp), and the primer extension reaction mediates the transfer of the information of the coding tag to the record tag by using the spacer (Sp) as a primer site. The coding tag is shown as a duplex having a single-stranded spacer (Sp') sequence at one end away from the binder. This configuration minimizes the hybridization of the coding tag with internal sites in the record tag and is conducive to the hybridization of the terminal spacer (Sp) sequence of the record tag with the single-stranded spacer protrusion (Sp') of the coding tag. In addition, the extended record tag can be pre-annealed with an oligonucleotide (complementary to the encoder and spacer sequence) to prevent the hybridization of the coding tag with internal record tag sequence elements. Figure 5 B shows the final extended transcript tag generated after "n" binding cycles ("***" indicates intervening binding cycles not shown in the extended transcript tag) and the transfer of the encoded tag information and addition of a universal priming site at the 3'-end.

[0266] Figure 6 The transfer of coded tag information to an extended record tag by enzyme ligation is shown. Two different macromolecules and their respective record tags are shown, wherein the record tag extension is performed in parallel. The ligation can be facilitated by designing a double-stranded coded tag so that the spacer sequence (Sp) has a "sticky end" protrusion that anneals to a complementary spacer sequence (Sp') on the record tag. The complementary strand of the double-stranded coded tag transfers information to the record tag. When ligation is used to extend the record tag, the direction of extension can be 5' to 3' as shown, or optionally 3' to 5'.

[0267] Figure 7 A "spaceless" method is shown in which the coding tag information is transferred to the recording tag by chemical connection to connect the 3' nucleotide of the recording tag or extended recording tag to the 5' nucleotide of the coding tag (or its complement) without inserting a spacer sequence into the extended recording tag. The directions of the extended recording tag and the coding tag can also be converted to facilitate connecting the 5' end of the recording tag to the 3' end of the coding tag (or complement). In the example shown, hybridization between the complementary "auxiliary" oligonucleotide sequence ("recording auxiliary") on the recording tag and the coding tag is used to stabilize the complex to promote specific chemical connection of the recording tag to the complementary chain of the coding tag. The generated extended recording tag lacks a spacer sequence. A "click chemistry" version that can use chemical connection of DNA, PNA or similar nucleic acid polymers (for example, using azide and alkyne groups (shown as triplet symbols)) is also shown.

[0268] Figure 8 AB shows an exemplary method for writing a peptide post-translational modification (PTM) into an extended record tag before the N-terminal amino acid is degraded. Figure 8 A: A binding agent containing a coding tag having identification information about the binding agent (e.g., a phosphotyrosine antibody containing a coding tag having identification information of a phosphotyrosine antibody) can bind to the peptide. If phosphotyrosine is present in the peptide labeled with the recording tag, as shown in the figure, after the phosphotyrosine antibody binds to phosphotyrosine, the coding tag and the recording tag anneal through a complementary spacer sequence and the coding tag information is transferred to the recording tag to generate an extended recording tag. Figure 8 B: The extended record tag may contain the initial amino acid sequence (e.g. "aa 1 ”,“aa 2 ”,“aa 3 ”,…,“aa N ”) and the post-translational modification of the peptide (e.g. “PTM 1 ”,“PTM 2 ”)’s encoded tag information.

[0269] Fig. 9AB shows a plurality of cycles of binding of a binder to a macromolecule and transferring the information of a coded tag attached to the binder to a single record tag in a plurality of record tags, thereby generating a plurality of extended record tags that collectively represent the macromolecule, the plurality of record tags being co-localized at one site of a single macromolecule, the single macromolecule being attached to a solid support (e.g., a bead). In this figure, for example purposes only, the macromolecule is a peptide, and each cycle involves binding of a binder to an N-terminal amino acid (NTAA), recording a binding event by transferring the coded tag information to the record tag, and then removing the NTAA to expose a new NTAA. Fig. 9 A shows a plurality of recording tags (comprising a universal forward primer sequence and a UMI) co-localized with the macromolecule on a solid support. The individual recording tags have a common spacer sequence (Sp) complementary to the common spacer sequence in the coding tag of the binder, which can be used to initiate an extension reaction to transfer the coding tag information to the recording tag. Fig. 9 B shows a different pool of cycle-specific NTAA binders used for each successive binding cycle, each pool having a cycle-specific spacer sequence.

[0270] Fig.10 AC shows an example model of multiple cycles involving transferring information from a coding tag attached to a binding agent to a single recording tag among a plurality of recording tags, thereby generating a plurality of extended recording tags that collectively represent the macromolecule, wherein the plurality of recording tags are co-localized at one site of a single macromolecule, the single macromolecule being attached to a solid support (e.g., a bead). In this figure, for example purposes only, the macromolecule is a peptide, and each cycle process involves binding to an N-terminal amino acid (NTAA), recording a binding event, and then removing the NTAA to expose a new NTAA.

[0271] Fig.10 A shows multiple record tags (comprising a universal forward primer sequence and a UMI) co-localized with the macromolecule on a solid support, preferably one single molecule per bead. The individual record tags have different spacer sequences at their 3'-ends, which have different "cycle-specific" sequences (e.g., C 1 ,C 2 ,C 3 ,…C n ). Preferably, the record tags on each bead share the same UMI sequence. In the first binding cycle (cycle 1), multiple NTAA binders are contacted with the macromolecule. The binders in cycle 1 have a common 5'-spacer sequence (C'1) that is identical to the cycle 1C'1 of the record tags. 1 The binders in cycle 1 also have a common 3'-spacer sequence (C' 2), which is the same as the cycle 2 spacer C 2 During binding cycle 1, the first NTAA binder binds to the free N-terminus of the macromolecule and the information of the first coding tag is transmitted from the hybridized C 1 The primer extension of the sequence to the complementary C'1 spacer sequence is transferred to the homologous record tag. After removing the NTAA to expose the new NTAA, the binding cycle 2 brings multiple NTAA binders into contact with the single molecule, and the NTAA binders have a 5'-spacer sequence (C'2) of cycle 2 that is the same as the 3'-spacer sequence of cycle 1 and a common 3'-spacer sequence (C'3) of cycle 3. The second NTAA binder binds to the N-terminus of the macromolecule, and the information of the second coding tag is transferred to the homologous record tag by primer extension of the complementary C2 and C'2 spacer sequences. These cycles are repeated to "n" binding cycles, where the last extended record tag is capped with a universal reverse priming sequence to produce multiple extended record tags co-localized with a single macromolecule, where each extended record tag has the coding tag information from one binding cycle. Because each set of binders used in each consecutive binding cycle has a cycle-specific spacer sequence in the coding tag, the binding cycle information can be associated with the binder information in the generated extended record tag. Fig.10 B shows a different pool of cycle-specific NTAA binders used for each successive binding cycle, each pool having a cycle-specific spacer sequence. Fig.10 C shows how a collection of extended record tags co-localized at sites on the macromolecule are sequentially assembled using cycle-specific spacer sequences based on PCR, thereby providing an ordered sequence of the macromolecule. In a preferred model, multiple copies of each extended record tag are generated by amplification prior to cascade.

[0272] Fig.11AB shows that information is transferred from a recording tag to a coding tag or di-tag construct. Two methods of recording binding information are shown in (A) and (B). The binding agent can be any type of binding agent described herein; anti-phosphotyrosine binding agents are shown for illustration purposes only. For extended coding tags or di-tag constructs, instead of transferring the binding information in the coding tag to the recording tag, the information is transferred from the recording tag to the coding tag to generate an extended coding tag (A), or the information is transferred from the recording tag and the coding tag to a third di-tag-forming construct (B). The di-tag and extended coding tag contain the information of the recording tag (including a barcode, an optional UMI sequence, and an optional compartment tag (CT) sequence (not shown)) and the coding tag. The di-tag and extended coding tag can be eluted, collected, and optionally amplified and read out on a next-generation sequencer from the recording tag.

[0273] Fig.12 AD shows the design of PNA combined barcode / UMI record labeling and di-tag detection of binding events. Fig.12 In A, the construction of combinatorial PNA barcodes / UMIs by chemical ligation of four basic PNA word sequences (A, A'-B, B'-C, and C') is shown. Hybridized DNA arms are included therein to generate a spacer-free combinatorial template for combinatorial assembly of PNA barcodes / UMIs. Chemical ligation is used to stitch the annealed PNA "words" together. Fig.12 B shows a method for transferring the PNA information of the recording tag to the DNA intermediate. The DNA intermediate is capable of transferring information to the coding tag. That is, the complementary DNA word sequence is annealed with the PNA and chemically ligated (enzymatic ligation is optional if a ligase using the PNA template is found). Fig.12 In C, the DNA intermediate is designed to interact with the coding tag via a spacer sequence, SP. The strand displacement primer extension step displaces the attached DNA and transfers the record tag information from the DNA intermediate to the coding tag to produce an extended coding tag. A terminator nucleotide may be incorporated into the end of the DNA intermediate to prevent the coding tag information from being transferred to the DNA intermediate via primer extension. Fig.12 D: Alternatively, information can be transferred from the coding tag to the DNA intermediate to produce a di-tag construct.Terminator nucleotides can be incorporated into the ends of the DNA intermediate to prevent transfer of record tag information from the DNA intermediate to the coding tag.

[0274] Fig.13AE shows the protein component partitioning on the compartment barcode beads, followed by di-tag assembly by emulsion fusion PCR to generate a library of elements representing the peptide sequence composition. The amino acid content of the peptide can then be characterized by N-terminal sequencing or by attachment (covalent or non-covalent) of amino acid-specific chemical tags or binders associated with the encoding tag. The encoding tag contains a universal priming site, as well as a barcode, compartment label, and amino acid UMI for the amino acid identification. After information transfer, the ditags are mapped back to the original molecule by recording the tag UMI. Fig.13 In A, the proteome is split into droplets by barcoded beads. Peptides with associated record tags (including compartment barcode information) are attached to the surface of the beads. The liquid emulsion is broken to release the barcoded beads with partitioned peptides. Fig.13 In B, specific amino acid sites on the peptide are chemically labeled with a DNA-encoded tag covalently linked to a site-specific tag group. The DNA-encoded tag contains amino acid barcode information and optional amino acid UMI. Fig.13 C: The labeled peptide-record tag complex is released from the beads. Fig.13 D: The labeled peptide-recording tag complex is emulsified into a nano- or microemulsion such that there is on average less than one peptide-recording tag complex per compartment. Fig.13 E: Emulsion fusion PCR transfers the recorded tag information (eg, compartment barcode) to all of the DNA-encoded tags attached to amino acid residues.

[0275] Fig.14 Shown is the generation of an extended coding tag from an emulsified peptide record tag-coding tag complex. Fig.13The peptide complex of C is co-emulsified with PCR reagents to form droplets with a single peptide complex per droplet on average. The record tag associated with the peptide is amplified using a three-primer fusion PCR method, the amplified record tag is fused to multiple binder coding tags or covalently labeled amino acid coding tags, the coding tag is extended by primer extension to transfer the compartment tag information of the peptide UMI and the record tag to the coding tag, and the resulting extended coding tag is amplified. Each droplet has multiple extended coding tag types, and each amino acid coding sequence-UMI coding tag has a different extended coding tag type. In this way, both the identity and number of amino acids in the peptide can be determined. The U1 universal primer and the Sp primer are designed to have a higher melting Tm than the U2tr universal primer. This enables a two-step PCR, in which the first few cycles are performed at a higher annealing temperature to amplify the record tag, and then to a lower Tm so that the record tag and the coding tag are mutually guided during PCR to produce an extended coding tag, and the U1 and U2tr universal primers are used to initiate the amplification of the resulting extended coding tag product. In some embodiments, the synthetase extension from the U2tr primer can be prevented by using a photolabeled 3' blocking group (Young et al., 2008, Chem. Commun (Camb) 4: 462-464). tr After initiating extension of the coding tag on the amplified Sp' sequence of the recording tag, the U2 tr The 3' blocking group is removed and a higher temperature PCR is initiated to employ U1 and U2 tr Primers amplify the extended encoding tag.

[0276] Fig.15The use of proteome regions and barcodes is illustrated to help enhance the mappability and phasing of proteins. In peptide sequencing, proteins are usually digested into peptides. In this process, the information of the relationship between each peptide derived from the parent protein molecule and their relationship with the parent protein molecule is lost. In order to rebuild this information, each peptide sequence is mapped back to a set of protein sequences that may be their source. Using short and / or partial peptide sequences, and as the size and complexity (e.g., proteome sequence complexity) of the set increase, the task of finding specific matches in such a group becomes more difficult. The proteome is partitioned into barcoded (e.g., compartment-labeled) compartments or partitions, and then the protein is digested into peptides, and the compartment tags are connected to the peptides to reduce the "protein" space that the peptide sequence needs to be mapped, greatly simplifying the task in the case of complex protein samples. Using unique molecular identifiers (UMI) to label proteins before digestion into peptides helps peptides map back to the original protein molecules and allows annotation of phase information between post-translational modification (PTM) variants derived from the same protein molecule and identification of single protein (proteoforms) forms. Fig.15 A shows an example of proteome partitioning, which involves labeling a protein with a memory tag containing a partition barcode and subsequently fragmenting it into memory tag-labeled peptides. Fig.15 B: For partial peptide sequence information or even just composition information, the mapping is highly degenerate. However, pairing partial peptide sequence or composition information with information from multiple peptides of the same protein allows unique identification of the original protein molecule.

[0277] Fig.16 An exemplary pattern of compartment-labeled bead sequence design is shown. The compartment labels include X 5-20 barcodes to identify individual compartments and N 5-10 A unique molecular identifier (UMI) is used to identify the peptide to which the compartment tag is attached, where X and N represent degenerate nucleic acid bases or nucleobase words. The compartment tag can be single-stranded (upper depiction) or double-stranded (lower depiction). Optionally, the compartment tag can be a chimeric molecule comprising a peptide sequence having a recognition sequence for a protein ligase (e.g., butelase I) for attachment to a target peptide (left figure). Alternatively, a chemical group can be included in the compartment tag for pairing to a target peptide (e.g., an azide, as shown in the right figure).

[0278] Fig.17AB illustrates: (A) multiple extended record tags representing multiple peptides; (B) exemplary methods for target peptide enrichment by standard hybrid capture techniques. For example, hybrid capture enrichment can use one or more biotinylated "bait" oligonucleotides that hybridize with extended record tags representing one or more peptides of interest ("target peptides"), the one or more peptides of interest being derived from a library of extended record tags representing a peptide library. The bait oligonucleotide: target extended record tag hybrid pair is spun down from the solution through the biotin tag after hybridization to produce an enriched breakdown product of extended record tags representing peptides or target peptides. Separation ("pull-down") of the extended record tags can be accomplished, for example, using streptavidin-coated magnetic beads. The biotin group binds to the streptavidin on the magnetic beads, and separation is accomplished by immobilizing the magnetic beads using a magnet while removing or replacing the solution. Non-biotinylated competitor enrichment oligonucleotides that competitively hybridize with extended record tags representing unwanted or overabundant peptides can optionally be included in the hybridization step of the hybrid capture assay to adjust the amount of enriched target peptides. The non-biotinylated competitor oligonucleotide competes for hybridization to the target peptide, but due to the absence of the biotin group, the hybridized duplex is not captured during the capture step. Therefore, the enriched extended record label fraction can be adjusted by adjusting the ratio of competitor oligonucleotide to biotinylated "bait" oligonucleotide over a large dynamic range. This step is very important to solve the dynamic range problem of protein abundance in the sample.

[0279] Fig.18 AB shows an exemplary method of partitioning a single cell and a large number of protein groups into a single droplet, each droplet comprising a bead having a plurality of compartment tags attached thereto to associate peptides with their originating protein complexes or proteins derived from a single cell. The compartment tags comprise barcodes. Manipulation of droplet components after droplet formation: (A) single cells are partitioned into a single droplet, followed by cell lysis to release the cell proteome, and proteolysis to digest the cell proteome into peptides, and inactivate the protease after sufficient proteolysis; (B) a large number of proteomes are partitioned into multiple droplets, wherein a single droplet comprises a protein complex, followed by proteolysis to digest the protein complex into peptides, and inactivate the protease after sufficient proteolysis. After photo-release of photo-caged divalent cations, the encapsulated proteins can be digested into peptides with a heat-labile metalloprotease to activate the protease. The protease can be heat-inactivated after sufficient proteolysis, or the divalent cation can be chelated. The droplets contain hybridized or releasable compartment tags comprising a nucleic acid barcode (separate from the recording tag) that can be attached to the N- or C-terminal amino acid of the peptide.

[0280] Fig.19 AB shows an exemplary method for partitioning single cells and a large number of protein groups into individual droplets, each droplet comprising beads having multiple bifunctional record tags and compartment tags attached to the record tags to associate peptides with their original proteins or protein complexes, or to associate proteins with their origins. The compartment tags comprise barcodes. Operations of droplet components after droplet formation: (A) single cells are partitioned into individual droplets, followed by cell lysis to release the cell proteome, and proteolysis to digest the cell proteome into peptides, and inactivate the proteases after sufficient proteolysis; (B) a large number of proteomes are partitioned into multiple droplets, wherein a single droplet contains a protein complex, followed by proteolysis to digest the protein complex into peptides, and inactivate the proteases after sufficient proteolysis. In the presence of light caged divalent cations (e.g., Zn 2+ ) can be digested into peptides using heat-labile metalloproteinases. The proteases can be heat-inactivated after sufficient proteolysis, or the divalent cations can be chelated. The droplets contain hybrid or releasable compartment tags that contain a nucleic acid barcode (separate from the recording tag) that can be attached to the N- or C-terminal amino acid of the peptide.

[0281] Figure 20A-L shows the generation of compartment barcoded record labels attached to peptides. Compartment barcode technology (e.g., barcoded beads in microfluidic droplets, etc.) can be used to deliver compartment-specific barcodes to molecular contents encapsulated in specific compartments. (A) In a specific embodiment, the protein molecule is denatured, and the ε-amine group of the lysine residue (K) is chemically conjugated to an activated universal DNA tag molecule (comprising a universal trigger sequence (U1)), shown with an NHS group at the 5' end). After the universal DNA tag is conjugated to the polypeptide, excess universal DNA tags are removed. (B) The universal DNA marker polypeptide is hybridized with a nucleic acid molecule bound to a bead, wherein the nucleic acid molecule bound to a single bead comprises a group of unique compartment tag (barcode) sequences. Compartmentalization occurs by separating the sample into different physical compartments, such as droplets (represented by dotted ellipses). Alternatively, compartmentalization can be accomplished directly by immobilizing the labeled polypeptide on the bead surface, for example, by annealing a universal DNA tag on the polypeptide to the compartment DNA tag on the bead without the need for additional physical separation. A single polypeptide molecule interacts only with a single bead (e.g., a single polypeptide does not span multiple beads). However, multiple polypeptides can interact with the same bead. In addition to the compartment barcode sequence (BC), the nucleic acid molecule bound to the bead can consist of a common Sp (spacer) sequence, a unique molecular identifier (UMI), and a sequence complementary to the polypeptide DNA tag, U1'. (C) After annealing of the universal DNA-tagged polypeptide to the compartment tag bound to the bead, the compartment tag is released from the bead by cleaving the accessory linker. (D) Extending the annealed U1 DNA tag primer by polymerase-based primer extension using the compartment tag nucleic acid molecule derived from the bead as a template. The primer extension step as described in (C) can be performed after the compartment tag is released from the bead, or optionally, while the compartment tag is still attached to the bead (not shown). This effectively writes the barcode sequence on the compartment tag on the bead to the U1 DNA tag sequence on the polypeptide. This new sequence constitutes the recording tag. After primer extension, a protease, such as Lys-C (cleaves the C-terminus of lysine residues), Glu-C (cleaves the C-terminus of glutamate residues and to a lesser extent the C-terminus of glutamate residues), or a random protease such as proteinase K, is used to cleave the polypeptide into peptide fragments. (E) Each peptide fragment is labeled with an extended DNA tag sequence, which constitutes a recording tag on its C-terminal lysine for downstream peptide sequencing in the present disclosure. (F) The recording-tagged peptide is coupled to the azidated beads via a tensioned alkyne label, DBCO. The azidated beads optionally also contain a capture sequence complementary to the recording tag to promote the efficiency of DBCO-azide immobilization.It should be noted that removal of the peptides from the original beads and re-immobilization to new solid supports (e.g., beads) allows for optimal intermolecular spacing between the peptides, facilitating the peptide sequencing methods disclosed herein. In addition to using click chemistry to conjugate DNA tags with alkyne pre-labeled polypeptides (e.g., Figure 2B 20G-L illustrate similar concepts as shown in FIG20A-F. The azide and mTet chemistries are orthogonal, allowing click conjugation to DNA tags and click conjugation of iEDDA (mTet and TCO) to the sequencing substrate.

[0282] Fig.21 An exemplary method using a flow focusing T-junction for single cells and compartmentalization using beads for compartment labeling (e.g., barcoding) is shown. With two streams, cell lysis and protease activation (Zn 2+ mix).

[0283] Fig. 22 AB shows exemplary labeling details. (A) Compartment tags (DNA-peptide chimeras) are attached to the peptides by peptide ligation using Butelase I. (B) Before starting peptide sequencing, the tag information is transferred to the associated record tag. Optionally, the endopeptidase AspN, which selectively cleaves peptide bonds N-terminal to aspartic acid residues, can be used to cleave the compartment tag after the information is transferred to the record tag.

[0284] Fig.23AC Array-based barcodes for spatial proteomics-based tissue section analysis. (A) An array of a set of spatially encoded DNA barcodes (feature barcodes represented by BCij) is combined with tissue sections (FFPE or frozen). In one embodiment, the tissue sections are fixed and permeabilized. In a preferred embodiment, the array feature size is smaller than the cell size (~10 μm for human cells). (B) The arrayed tissue sections are treated with a reagent to reverse crosslinks (e.g., by an antigen retrieval protocol using citraconic anhydride (Namimatsu, Ghazizadeh et al., 2005), and the proteins therein are then labeled with site-reactive DNA, thereby effectively labeling all protein molecules with DNA record tags (e.g., lysine tags, released after antigen retrieval). After labeling and washing, the array-bound DNA barcode sequences are cleaved and diffused into the arrayed tissue sections and hybridized with the DNA record tags attached to the proteins therein. (C) The arrayed tissue is now subjected to polymerase extension to transfer the information of the hybridized barcode to the DNA record tags that label the proteins. After the barcode information is transferred, the arrayed tissue is scraped off the slide, optionally digested with a protease, and the proteins or peptides are extracted into a solution.

[0285] Fig.24A -B shows two different exemplary DNA target macromolecules (AB and CD), which are fixed on beads and measured by binding agents connected to coding tags. This model system is used to demonstrate the single-molecule behavior of coding tags transferred from binding agents to proximal reporter tags. In a preferred embodiment, the coding tags are integrated into the extended recording tags by primer extension. Fig.24A The interaction of the AB macromolecule with the A-specific binder ("A", which is an oligonucleotide sequence complementary to the "A" component of the AB macromolecule) and the transfer of the information of the associated coding tag to the recording tag through primer extension, as well as the transfer of the information of the B-specific binder ("B", which is an oligonucleotide sequence complementary to the "B" component of the AB macromolecule) and the associated coding tag to the recording tag through primer extension are shown. The coding tags A and B have different sequences, and for ease of identification in this illustration, they also have different lengths. The different lengths facilitate analysis of the coding tag transfer by gel electrophoresis without the need for analysis by next-generation sequencing. The binding of the A' and B' binders is shown as an alternative possibility for a single binding cycle. If a second cycle is added, the extended recording tag will be further extended. Depending on which of the A' or B' binders is added in the first and second cycles, the extended recording tag can contain coding tag information in the form of AA, AB, BA, and BB. Therefore, the extended recording tag contains information about the order of binding events and the identity of the binders. Similarly, Fig. 24BDemonstrates the interaction of a CD macromolecule with a C-specific binder ("C", an oligonucleotide sequence complementary to the "C" component of the CD macromolecule) and the transfer of the information of the associated coding tag to the recording tag by primer extension, and a D-specific binder ("D", an oligonucleotide sequence complementary to the "D" component of the CD macromolecule) and the transfer of the information of the associated coding tag to the recording tag by primer extension. Coding tags C and D have different sequences and, for ease of identification, also have different lengths in this illustration. The different lengths facilitate analysis of coding tag transfer by gel electrophoresis without the need for analysis by next-generation sequencing. The binding of C' and D' binders is shown as an alternative possibility for a single binding cycle. If a second cycle is added, the extended recording tag will be further extended. Depending on which of the C' or D' binders is added in the first and second cycles, the extended recording tag can contain coding tag information in the form of CC, CD, DC, and DD. The coding tag can optionally contain a UMI. The inclusion of a UMI in the coding tag can record additional information of the binding event; and can distinguish binding events at the level of a single binder. This can be useful if a single binder can participate in more than one binding event (e.g., its binding affinity is such that it can dissociate and rebind frequently enough to participate in more than one event). It can also be used for error correction. For example, in some cases the encoding tag may transfer information to the recording tag two or more times in the same binding cycle. Using a UMI can expose those repeated information transfer events that may all be associated with a single binding event.

[0286] Fig.25 An exemplary DNA target macromolecule (AB) is shown and fixed on a bead, and is measured by a binder connected to a coding tag. A specific binder ("A", an oligonucleotide complementary to the A component of the AB macromolecule) interacts with the AB macromolecule and transfers the information of the relevant coding tag to the recording tag by connection. B specific binder ("B", an oligonucleotide complementary to the B component of the AB macromolecule) interacts with the AB macromolecule and transfers the information of the relevant coding tag to the recording tag by connection. Coding tags A and B have different sequences and, for ease of identification, also have different lengths in this illustration. The different lengths facilitate analysis of coding tag transfer by gel electrophoresis without the need for analysis by next generation sequencing.

[0287] Fig.26 AB show exemplary DNA-peptide macromolecules for binding / encoding tag transfer by primer extension. Fig.26A shows an exemplary oligonucleotide-peptide target macromolecule ("A" oligonucleotide-CMyc peptide) immobilized on a bead. A cMyc-specific binding agent (e.g., an antibody) interacts with the cMyc peptide portion of the macromolecule, and the information of the associated coding tag is transferred to the recording tag. The transfer of the information of the cMyc coding tag to the recording tag can be analyzed by gel electrophoresis. Fig.26 B shows an exemplary oligonucleotide-peptide target macromolecule ("C" oligonucleotide-hemagglutinin (HA) peptide) immobilized on beads. An HA-specific binding agent (e.g., an antibody) interacts with the HA peptide portion of the macromolecule, and the information of the associated coding tag is transferred to the recording tag. The transfer of the information of the coding tag to the recording tag can be analyzed by gel electrophoresis. The combination of cMyc antibody-coding tag and HA antibody-coding tag is demonstrated as an alternative possibility for a single binding cycle. If a second binding cycle is performed, the extended recording tag will be further extended. Depending on which of the cMyc antibody-coding tag or HA antibody-coding tag is added in the first and second binding cycles, the extended recording tag may contain coding tag information in the form of cMyc-HA, HA-cMyc, cMyc-cMyc and HA-HA. Although not shown, additional binding agents may also be introduced to enable detection of the A and C oligonucleotide components of the macromolecule. Thus, hybrid macromolecules comprising different types of backbones can be analyzed by transferring information to a record tag, which contains information about the sequence of binding events as well as the identity of the binding agent, and reading out the extended record tag.

[0288] Fig.27A-D. Generation of error-correcting barcodes. (A) A subset of 65 error-correcting barcodes (SEQ ID NOs: 1-65) was selected from 77 barcodes derived from the R package 'DNABarcodes' (https: / / bioconductor.riken.jp / packages / 3.3 / bioc / manuals / DNAB arcodes / man / DNABarcodes.pdf) using the recommended parameters [create.dnabarcodes(n=15, dist=10)]. The algorithm generates 15-mer "Hamming" barcodes that can correct substitution errors to a distance of four substitutions and detect errors to nine substitutions. The subset of 65 barcodes was created by filtering out those barcodes that did not show a variety of nanopore current levels (for nanopore-based sequencing) or were too correlated with other members of the group. (B) Schematic diagram of the predicted nanopore current levels of 15-mer barcodes passing through the nanopore. The predicted current was calculated by dividing each 15-mer barcode word into a composite group of 11 overlapping 5-mer words, and the corresponding current level was predicted using the 5-mer R9 nanopore current level lookup table (template_median68pA.5mers.model (https: / / github.com / jts / nanopolish / tree / master / etc / r9-models) as the barcode passed through the nanopore one base at a time. As can be understood from (B), this set of 65 barcodes exhibits unique current characteristics for each of its members. (C) The generation of PCR products of extended record tags as a model for nanopore sequencing shows the use of overlapping DTR sets and DTR primers. The PCR amplicons are then connected to form a cascaded extended record tag model. (D) Generation of exemplary "extended record tag" model nanopore sequencing reads (read length 734 bases) as shown Fig.27C The quality score of the MinIon R9.4 Read is 7.2 (poor read quality). However, even with poor read quality (Qscore = 7.2), the barcode sequence can be easily identified using lalign. The 15-mer spacer element is underlined. The barcode can be aligned in forward or reverse orientation, indicated by BC or BC'.

[0289] Fig.28AD. Analyte-specific labeling of proteins using DNA tags. (A) A binder targeting a target protein analyte in its native conformation comprises an analyte-specific barcode (BCA) hybridized to a complementary analyte-specific barcode (BCA') on a DNA tag. Alternatively, the DNA tag may be attached to the binder via a cleavable linker, and the DNA tag is directly "clicked" onto the protein and then cleaved from the binder (via the cleavable linker). The DNA tag comprises a reactive coupling group (e.g., a click chemistry reagent (e.g., azide, mTet, etc.) for coupling the target protein and other functional components (e.g., a universal primer sequence (Sp)), a sample barcode (BCS), an analyte-specific barcode (BCS), and a DNA tag (BCS). A ) and spacer sequence (Sp). Sample barcodes (BCS) can also be used to label and distinguish proteins from different samples. The DNA recording tag can also include an orthogonal coupling group (e.g., mTet) for subsequent coupling to a substrate surface. For click chemistry coupling of the recording tag to the target protein, the protein is pre-labeled with a click chemistry coupling group that is homologous to the click chemistry coupling group on the DNA recording tag (e.g., the alkyne group on the protein is homologous to the azide group on the DNA recording tag). Examples of reagents for labeling DNA recording tags with coupling groups for click chemistry coupling include alkyne-NHS reagents for lysine labeling, alkyne-benzophenone reagents for photoaffinity labeling, and the like. (B) After the binding agent binds to the proximal target protein, the reactive coupling group (e.g., azide) on the recording tag is covalently linked to the homologous click chemistry coupling group (shown as a three-line symbol) on the proximal protein. (C) After labeling the target protein analyte with the recording tag, the protein is pre-labeled with a uracil-specific excision reagent (e.g., USER TM ) digests uracil (U) to remove the connected binding agent. (D) The target protein analyte labeled with the DNA record tag is fixed to the surface of the matrix using a suitable bioconjugation chemical reaction, such as click chemistry (alkyne-azide binding pair, methyl tetrazine (mTET)-trans-cyclooctene (TCO) binding pair, etc.). In certain embodiments, the entire target protein-record tag labeling assay is performed in a single tube containing many different target protein analytes using a binding agent library and a record tag library. After the protein analytes in the sample are targeted and labeled with a record tag containing a sample bar code (BCS), multiple protein analyte samples can be aggregated before the fixation step in (D). Therefore, in certain embodiments, up to thousands of protein analytes in hundreds of samples can be labeled and fixed in a single tube next generation protein assay (NGPA), greatly saving expensive affinity reagents (such as antibodies).

[0290] Fig.29 AE. DNA record label and polypeptide conjugation. (A) Denatured polypeptides are labeled with bifunctional click chemistry reagents, such as alkyne-NHS ester (acetylene-PEG-NHS ester) reagents or alkyne dibenzophenones to produce alkyne-tagged (triple line symbol) polypeptides. Alkynes can also be strained alkynes, such as cyclooctyne, including dibenzocyclooctyl (DBCO) and the like. (B) An example of a DNA record label design chemically coupled to the alkyne-tagged polypeptide is shown. The record label includes a universal primer sequence (P1), a barcode (BC) and a spacer sequence (Sp). The record label is labeled with an mTet group to couple to a substrate surface and with an azide group for coupling to an alkyne group of the labeled polypeptide. (C) Denatured alkyne-tagged proteins or polypeptides are labeled with record labels via alkyne and azide groups. Optionally, the record label-tagged polypeptide can be further labeled with a compartment barcode, such as by annealing with a complementary sequence connected to a compartment bead and primer extension (also known as polymerase extension), or as shown in Figures 20H-J. (D) Protease digestion of the record tag-labeled polypeptide produces a group of record tag-labeled peptides. In some embodiments, some peptides will not be labeled by any record tag. In other embodiments, some peptides can be attached with one or more record tags. (E) The record tag-labeled peptide is fixed on the substrate surface using the reverse electron Diels-Alder (iEDDA) click chemistry reaction between the substrate surface functionalized with the TCO group and the mTet group of the record tag connected to the peptide. In certain embodiments, a cleaning step can be used between the different stages shown. The use of orthogonal click chemistry (e.g., azide-alkyne and mTet-TCO) allows the polypeptide to be click-labeled with a record tag, and the record tag-labeled peptide is fixed to the substrate surface with click chemistry (see, McKay et al., 2014, Chem.Biol.21:1075-1101, the entire contents of which are incorporated herein by reference).

[0291] Fig.30AE. Write the sample barcode to the record tag after the initial labeling of the DNA tag of the polypeptide. (A) Label the denatured polypeptide with a bifunctional click chemistry reagent such as an alkyne-NHS reagent or alkyne benzophenone to produce an alkyne-labeled polypeptide. (B) After the polypeptide is labeled with alkyne (or an alternative click chemistry reagent), a DNA tag containing a universal primer sequence (P1) and labeled with an azide group and an mTet group is coupled to the polypeptide through an azide-alkyne interaction. It should be understood that other click chemistry interactions can be used. (C) A record tag DNA construct containing sample barcode information (BCS') and other record tag functional components (e.g., universal primer sequence (P1'), spacer sequence (Sp')) is annealed with the DNA tag-labeled polypeptide through a complementary universal priming sequence (P1-P1'). Transfer the record tag information to the DNA tag by polymerase extension. (D) Protease digestion of the record tag-labeled polypeptide produces a group of record tag-labeled peptides. (E) Using the inverse electron Diels-Alder (iEDDA) click chemistry reaction between the substrate surface functionalized with the TCO group and the mTet group of the record tag connected to the peptide, the peptide labeled with the record tag is fixed on the substrate surface. In certain embodiments, a cleaning step can be used between the different stages shown. The use of orthogonal click chemistry (e.g., azide-alkyne and mTet-TCO) allows the peptide to be click-chemically labeled with the record tag, and the peptide labeled with the record tag is fixed to the substrate surface with click chemistry (see, McKay et al., 2014, Chem.Biol.21:1075-1101, the entire contents of which are incorporated herein by reference).

[0292] Fig.31 AE. Bead compartmentalization for barcoded polypeptides. (A) Polypeptides are labeled in solution using heterobifunctional click chemistry reagents using standard bioconjugation or photoaffinity labeling techniques. Possible labeling sites include the ε-amine of lysine residues (e.g., using NHS-alkyne as shown) or the carbon backbone of the peptide (e.g., using benzophenone-alkyne). (B) An azide-labeled DNA tag comprising a universal primer sequence (P1) is coupled to the alkyne group of the labeled polypeptide. (C) The DNA-tagged polypeptide is annealed to a DNA-tagged bead via complementary DNA sequences (P1 and P1'). The DNA-tagged on the bead comprises a spacer sequence (Sp'), a compartmental barcode sequence (BC P'), an optional unique molecular identifier (UMI) and a universal sequence (P1'). The DNA record tag information is transferred to the DNA tag on the polypeptide by polymerase extension (or, ligation can be used). After the information transfer, the resulting polypeptide contains multiple record tags, which contain several functional elements including compartment barcodes. (D) Protease digestion of the record tag-labeled polypeptide produces a group of record tag-labeled peptides. The record tag-labeled peptides are dissociated from the beads and (E) re-immobilized on the sequencing matrix (for example, as shown in the figure, using iEDDA click chemistry between mTet and TCO groups).

[0293] Fig.32A-H. Example of workflow for next generation protein assay (NGPA). Protein samples are labeled with DNA record tags consisting of several functional units, such as a universal reference sequence (P1), a barcode sequence (BC), an optional UMI sequence, and a spacer sequence (Sp) (capable of binding to a reagent encoding tag to facilitate information transfer). (A) The labeled protein is immobilized (passively or covalently) on a matrix (e.g., beads, porous beads, or porous matrix). (B) The matrix is ​​blocked with protein, and a competitive oligonucleotide (Sp') complementary to the spacer sequence is optionally added to minimize non-specific interactions of the analyte record tag sequence. (C) Analyte-specific antibodies (attached with relevant encoding tags) are incubated with the matrix-bound protein. The encoding tag may contain uracil bases for subsequent uracil-specific cleavage. (D) After antibody binding, excess competitive oligonucleotide (Sp') (if added) is washed away. The encoding tag is transiently annealed to the record tag through the complementary spacer sequence, and the encoding tag information is transferred to the record tag in a primer extension reaction to produce an extended record tag. If the fixed protein is denatured, the bound antibody and annealed coding tag can be removed by washing under alkaline conditions, such as with 0.1N NaOH. If the fixed protein is in a native conformation, milder conditions may be required to remove the bound antibody and coding tag. Examples of milder antibody removal conditions are outlined in Figures EH. (E) After the information is transferred from the coding tag to the recording tag, the coding tag is cut (cut) at its uracil site using a uracil-specific excision reagent (e.g., USERTM) enzyme mixture. (F) The bound antibody is removed from the protein using a high salt, low / high pH wash solution. The truncated DNA coding tag retained on the antibody is very short and also elutes quickly. Longer DNA coding tag fragments may or may not remain annealed to the recording tag. (G) As in steps (B)-(D), the second binding cycle begins and the second primer extension step transfers the coding tag information from the second antibody to the extended recording tag by primer extension. (H) The result of two binding cycles is a cascade of the binding information from the first antibody attached to the recording tag and the binding information from the second antibody.

[0294] Fig.33AD: Single-step next-generation protein assay (NGPA) using multiple binders and enzyme-mediated sequential information transfer. An NGPA assay employs simultaneous binding of two homologous binders (e.g., antibodies) immobilized protein molecules. After multiple homologous antibody binding events, a combined primer extension and DNA nicking step is used to transfer the information of the coding tag from the bound antibody to the recording tag. The insertion symbol (^) in the coding tag represents a double-stranded DNA nicking endonuclease site. (A) In the illustrated embodiment, the coding tag of the antibody bound to epitope 1 (Epi#1) of the protein transfers the coding tag information (e.g., the encoder sequence) to the recording tag in a primer extension step following hybridization with a complementary spacer sequence. (B) Once the double-stranded DNA duplex is formed between the extended recording tag and the coding tag, a nicking endonuclease such as Nt.BsmAI, which is active at 37°C and only cuts single-stranded DNA on the double-stranded DNA matrix, is used to cut the coding tag. After the nicking step, the duplex formed by the truncated coding tag binder and the extended recording tag is thermodynamically unstable and dissociates. The longer coding tag fragment may or may not remain annealed to the recording tag. (C) This allows the coding tag from the antibody that binds to epitope #2 (Epi#2) of the protein to anneal to the extended recording tag through a complementary spacer sequence, and the extended recording tag is further extended by information transfer from the coding tag of the Epi#2 antibody to the extended recording tag via primer extension. (D) Again, after the double-stranded DNA duplex is formed between the extended recording tag and the coding tag of the Epi#2 antibody, the coding tag is cut by a nicking endonuclease such as Nb.BssSI.

[0295] In certain embodiments, it is preferred to use a non-strand displacement polymerase (also referred to as polymerase extension) during primer extension. The non-strand displacement polymerase prevents the cleaved coding tag short column from extending, which remains annealed to the record tag by more than one single base. The process of (A)-(D) can repeat itself until all of the coding tags of the proximal bound binding agents are "consumed" by the hybridization, information transfer to the extended record tag, and the cleavage step. The coding tag can include the same coding sequence as all binding agents (e.g., antibodies) specific for a given analyte (e.g., a homologous protein), can include an epitope-specific coding sequence, or can include a unique molecular identifier (UMI) to distinguish different molecular events.

[0296] Fig.34AC: Controlled density of recording labels - peptides are fixed using reactive groups on the titration substrate surface. (A) The density of peptides on the surface of the substrate can be titrated by controlling the density of functional coupling groups on the substrate surface. This can be achieved by derivatizing the substrate surface with an appropriate ratio of active coupling molecules and "virtual" coupling molecules. In the example shown, NHS-PEG-TCO reagent (active coupling molecule) is combined with NHS-mPEG (virtual molecule) in a determined ratio and TCO to derive the amine surface. Functionalized PEG has various molecular weights ranging from 300 to more than 40,000. (B) A bifunctional 5'amine DNA recording label (mTet is the other functional group) is coupled to the N-terminal Cys residue of the peptide using a succinimidyl 4-(N-maleimidomethyl) cyclohexane-1 (SMCC) bifunctional crosslinker. The internal mTet-dT group on the recording label is generated from the azide-dT group using mTetrazine-Azide. (C) The recording tag of the labeled peptide is immobilized on the activated substrate surface of (A) using iTDA click chemistry reaction with mTet and TCO. The mTet-TCO iEDDA coupling reaction is very fast, efficient and stable (mTet-TCO is more stable than Tet-TCO).

[0297] Fig.35AC. Next generation protein sequencing (NGPS) combined with cycle-specific encoding tags. (A) Design of NGPS assay using cycle-specific N-terminal amino acid (NTAA) binder encoding tags. The NTAA binder (e.g., an antibody specific for N-terminal DNP-labeled tyrosine) binds to the DNP-labeled NTAA of a peptide, which is associated with a record tag comprising a universal primer sequence (P1), a barcode (BC), and a spacer sequence (SP). When the binder binds to the cognate NTAA of the peptide, the encoding tag associated with the NTAA binder approaches the record tag and anneals with the record tag through a complementary spacer sequence. The encoding tag information is transferred to the record tag by primer extension. In order to track which binding cycle the encoding tag represents, the encoding tag may include a cycle-specific barcode. In certain embodiments, the encoding tag of the binder bound to the analyte has the same encoder barcode independent of the cycle number, which is combined with a unique binding cycle-specific barcode. In other embodiments, the encoding tag of the binder for the analyte comprises a unique encoder barcode for combined analyte binding cycle information. In either method, a common spacer sequence can be used to encode tags for binders in each binding cycle. (B) In this example, binders from each binding cycle have a short binding cycle-specific barcode to identify the binding cycle, which together with the encoder barcode that identifies the binder provides a unique combined barcode that identifies a particular binder-binding cycle combination. (C) After the binding cycle is completed, the extended record tag can be converted into an amplifiable library using a capping cycle step, where, for example, a cap comprising a universal primer sequence P1 and a spacer sequence Sp' linked to a universal primer sequence P2 is initially annealed to the extended record tag via complementary P1 and P1' sequences, bringing the cap into proximity with the extended record tag. Complementary Sp and Sp' sequences in the extended record tag and cap annealing and primer extension add the second universal primer sequence (P2) to the extended record tag.

[0298] Fig.36A-F. A DNA-based model system for demonstrating information transfer from coding tags to recording tags. Exemplary binding and intramolecular writing were demonstrated by an oligonucleotide model system. The targeting agents A' and B' in the coding tags are designed to hybridize with the target binding regions A and B in the recording tags. The recording tag (RT) mixture is prepared by pooling two recoding tags saRT_Abc_v2 (A target) and saRT_Bbc_V2 (B target) of the same concentration. The recording tag is biotinylated at its 5' end and contains a unique target binding region, a universal forward primer sequence, a unique DNA barcode, and an 8-base common spacer sequence (Sp). The coding tag contains a unique encoder barcode base flanked by an 8-base common spacer sequence (Sp'), one of which is covalently linked to the A or B target agent via a polyethylene glycol linker. (A) The biotinylated recording tag oligonucleotides (saRT_Abc_v2 and saRT_Bbc_V2) and the biotinylated Dummy-T10 oligonucleotide are fixed together on streptavidin-labeled beads. The record tag is designed with an A or B capture sequence (recognized by the cognate binders - A' and B', respectively) and a corresponding barcode (rtA_BC and rtB_BC) to identify the binding target. All barcodes in this model system were selected from the 65 15-mer barcode groups (SEQ ID NO: 1-65). In some cases, 15-mer barcodes were combined to form longer barcodes for easy gel analysis. In particular, rtA_BC = BC_1 + BC_2; rtB_BC = BC_3. Two encoding tags for binders homologous to the A and B sequences of the record tag were also synthesized, namely CT_A'-bc (encoder barcode = BC_5) and CT_B'-bc (encoder barcode = BC_5 + BC_6). Blocking oligonucleotides (DupCT_A'BC and DupCT_AB'BC) complementary to a portion of the coding tag sequence (leaving a single-stranded Sp' sequence) are optionally pre-annealed to the coding tag before the coding tag is annealed to the bead-fixed recording tag. A strand displacement polymerase removes the blocking oligonucleotide during polymerase extension. The barcode legend (inset) shows the 15-mer barcode assignment of the functional barcodes in the recording tag and the coding tag. (B) The recording tag barcode design and the coding tag encoder barcode design provide a simple gel analysis of the "intra-molecular" vs. "intermolecular" interactions between the recording tag and the coding tag. In this design, the gel products produced by the undesired "intermolecular" interactions (A recording tag with B' coding tag; and B recording tag with A' coding tag) are 15 bases longer or shorter than the desired "intra-molecular" (A recording tag with A' coding tag; B recording tag with B' coding tag) reaction products.The primer extension step changes the A' and B' encoding tag barcodes (ctA'_BC, ctB'_BC) to reverse complement barcodes (ctA_BC and ctB_BC). (C) Primer extension assay demonstrates information transfer from encoding tags to recording tags, and the addition of adapter sequences by primer extension on annealed EndCap oligonucleotides for PCR analysis. (D) "Intramolecular" information transfer is optimized by titrating the surface density of recording tags using Dummy-T20 oligonucleotides. Biotinylated recording tag oligonucleotides were mixed with biotinylated Dummy-T20 oligonucleotides in various ratios of 1:0, 1:10, up to 1:10000. At reduced recording tag density (1:10 3 and 1:10 4), "intramolecular" interactions dominate over "intermolecular" interactions. (F) As a simple extension of the DNA model system, although a simple protein binding system containing a Nano-Tag15 peptide-streptavidin binding pair (KD ~ 4nM) (Perbandt et al., 2007, Proteins 67: 1147-1153) is illustrated, any number of peptide binder model systems can be used. The Nano-Tag15 peptide sequence is (fM)DVEAWLGARVPLVET (SEQ ID NO: 131) (fM = Formyl-Met). The Nano-Tag15 peptide also contains a short, flexible linker peptide (GGGGS) and a cysteine ​​residue for coupling to the DNA record tag. Other example peptide tag-cognate binder pairs include: calmodulin binding peptide (CBP)-calmodulin (KD ~ 2pM) (Mukherjee et al., 2015, J. Mol. Biol. 427: 2707-2725), amyloid-β (Aβ16–27) peptide-US7 / Lcn2 anticalin (0.2nM) (Rauth et al., 2016, Biochem. J. 473: 1563-1578), PA tag / NZ-1 antibody (KD ~ 400pM), FLAG-M2Ab (28nM), HA-4B2 Ab (1.6nM) and Myc-9E10 Ab (2.2nM) (Fujii et al., 2014, Protein Expr. Purif. 95: 240-247). (E) As a test for the transfer of intramolecular information from the binding agent encoding tag to the recording tag by primer extension, oligonucleotide "binding agents" that bind to complementary DNA sequence "A" can be used for testing and development. This hybridization event is substantially stronger than fM affinity. Streptavidin can be used as a test binding agent for Nano-tag15 peptide epitopes. Although the peptide tag-binding agent interaction is highly affinity, it is easy to destroy it using acidic and / or high salt washing (Perbandt et al., supra).

[0299] Fig.37AB. Transfer of information from UMI-tagged N- or C-termini to DNA-tagged polypeptide bodies using nano- or microemulsion PCR. (A) A polypeptide is tagged at its N- or C-terminus with a nucleic acid molecule containing a unique molecular identifier (UMI). The UMI can be flanked by a sequence for priming subsequent PCR. The polypeptide is then "body-tagged" at its internal sites with a separate DNA tag containing a sequence complementary to the priming sequence flanking the UMI. (B) The resulting tagged polypeptide is emulsified and subjected to emulsion PCR (ePCR) (alternatively, an emulsion in vitro transcription-RT-PCR (IVT-RT-PCR) reaction or other suitable amplification reaction can be performed) to amplify the N- or C-terminal UMI. A microemulsion or nanoemulsion is formed so that the average droplet diameter is 50-1000 nm and there is less than one polypeptide per droplet on average. Snapshots of the droplet contents before and after PCR are shown in the left and right panels, respectively. The UMI amplicon hybridizes to the internal polypeptide body DNA tag through a complementary priming sequence, and the UMI information is transferred from the amplicon to the internal polypeptide body DNA tag through primer extension.

[0300] Fig.38 . Single cell proteomics. Encapsulate and lyse cells in droplets containing polymer forming subunits (e.g., acrylamide). Polymerize the polymer forming subunits (e.g., polyacrylamide) and crosslink proteins to the polymer matrix. Destroy the emulsion droplets and release polymer gel particles containing single cell protein lysates attached to the permeable polymer matrix. By including a denaturant such as urea in the lysis and encapsulation buffer, the protein is crosslinked to the polymer matrix in its native conformation or denatured state. Using many methods known in the art or disclosed herein, including emulsification or combinatorial indexing using barcode beads, a record tag containing a compartment barcode and other record tag components (e.g., a universal priming sequence (P1), a spacer sequence (Sp), an optional unique molecular identifier (UMI)) is attached to the protein. The polymer gel beads containing the single cell protein can also be protease digested after the addition of the record tag to produce record tag-labeled peptides suitable for peptide sequencing. In certain embodiments, the polymer matrix can be designed to dissolve in suitable additives, such as disulfide cross-linked polymers that are disrupted upon exposure to reducing agents such as tris(2-carboxyethyl)phosphine (TCEP) or dithiothreitol (DTT).

[0301] Fig.39AE. Enhancement of amino acid cleavage reactions using bifunctional N-terminal amino acid (NTAA) modifiers and chimeric cleavage reagents. (A) and (B) Peptides attached to a solid substrate are modified with a bifunctional NTAA modifier, such as biotin-phenylisothiocyanate (PITC). (C) Low affinity Edmanase (>μM Kd) is recruited to biotin-PITC labeled NTAA using a streptavidin-Edmanase chimeric protein. (D) The efficiency of the Edmanase cleavage is greatly improved due to the increase in local effective concentration caused by the biotin-streptavidin interaction. (E) The cleaved biotin-PITC labeled NTAA and the associated streptavidin-Edmanase chimeric protein diffuse after cleavage. Many other bioconjugate recruitment strategies can also be used. Azide-modified PITC is commercially available (4-azidophenylisothiocyanate, Sigma), and azide-PITC can be simply converted to other bioconjugates of PITC, such as biotin-PITC, by reaction with alkyne-biotin via click chemistry.

[0302] Figure 40A-I: Generation of C-terminal record tag-labeled peptides from protein lysates (can be encapsulated in gel beads). (A) The denatured polypeptide is reacted with anhydrides to label lysine residues. In one embodiment, the lysine is labeled with mTet using a mixture of alkyne (mTet)-substituted citraconic anhydride + propionic anhydride. (Shown as striped rectangles) (B) The result is an alkyne (mTet) labeled polypeptide in which a portion of the lysines are blocked with propionic acid groups (shown as squares on the polypeptide chain). The alkyne (mTet) group can be used for click chemistry based on DNA labeling. (C) DNA tags for alkyne or mTet groups (shown as solid rectangles) are attached by using azide or trans-cyclooctene (TCO) tags, respectively. (D) As Fig.31As shown in , a primer extension step is used to attach a barcode and functional elements such as a spacer (Sp) sequence and a universal priming sequence to a DNA tag to produce a record-labeled polypeptide. The barcode can be a sample barcode, a partition barcode, a compartment barcode, a spatial position barcode, etc., or any combination thereof. (E) The obtained record-labeled polypeptide is fragmented into a record-labeled peptide using a protease or chemical method. (F) For illustration, a peptide fragment labeled with two record labels is shown. (G) A DNA tag containing a universal primer sequence complementary to the universal primer sequence in the record label is connected to the C-terminus of the peptide. The C-terminal DNA tag also contains a group for conjugating the peptide to a surface. (H) The complementary universal primer sequence in the C-terminal DNA tag and the randomly selected record label are annealed. An intramolecular primer extension reaction is used to transfer information from the record label to the C-terminal DNA tag. (I) The internal record label on the peptide is coupled to a lysine residue by maleic anhydride, and the coupling is reversible at acidic pH. The internal recording tag is cleaved from the lysine residue of the peptide at acidic pH, leaving the C-terminal recording tag. The newly exposed lysine residue can optionally be blocked with a non-hydrolysable anhydride such as propionic anhydride.

[0303] Fig.41 .Workflow of a preferred embodiment of the NGPS assay.

[0304] Fig.42AD. Exemplary steps of NGPS sequencing analysis. The N-terminal amino acid (NTAA) acetylation or amidation step on the surface-bound peptide of the record tag label can occur before or after the NTAA binding agent is combined, depending on whether the NTAA binding agent has been engineered to bind acetylated NTAA or native NTAAs. In the first case, (A) the peptide is initially acetylated by chemical means using acetic anhydride or enzymatic acetylation using N-terminal acetyltransferase (NAT) at the NTAA. (B) The NTAA is recognized by NTAA binding agents such as engineered anticalins, aminoacyl tRNA synthetases (aaRS), ClpS, etc. The DNA encoding tag is attached to the binding agent and comprises a barcode encoding sequence that identifies the specific NTAA conjugate. (C) After the acetylated NTAA is combined by the NTAA binding agent, the DNA encoding tag is instantaneously annealed with the record tag by a complementary sequence, and the encoding tag information is transferred to the record tag by polymerase extension. In another alternative embodiment, the record tag information is transferred to the encoding tag by polymerase extension. (D) The acetylated NTAA is cleaved from the peptide by an engineered acyl peptide hydrolase (APH), which catalyzes the hydrolysis of the terminal acetylated amino acid from the acetylated peptide. After the acetylated NTAA is cleaved, the cycle repeats itself starting from the acetylation of the newly exposed NTAA. Although N-terminal acetylation is used as an exemplary mode of NTAA modification / cleavage, other N-terminal groups, such as guanyl groups, can be replaced by concomitant changes in the cleavage chemistry. If guanidinylation is used, the guanylated NTAA can be cleaved under mild conditions using a 0.5-2% NaOH solution (see Hamada, 2016, the entire contents of which are incorporated by reference). APH is a serine peptidase that can catalyze the removal of Nα-acetylated amino acids from closed peptides, and it belongs to the prolyl oligopeptidase (POP) family (clan SC, family S9). It is a key regulator of N-terminal acetylated proteins in eukaryotic cells, bacteria, and archaea.

[0305] Fig.43 AB: Exemplary record tag-coding tag design features. (A) Structure of an exemplary record tag-associated protein (or peptide) and a binder (e.g., anticalin) bound to the associated coding tag. Thymidine (T) bases are inserted between the spacer (Sp') and barcode (BC') sequences on the coding tag to accommodate random non-templated 3' end adenosine (A) addition in the primer extension reaction. (B) DNA coding tags are linked to binders (e.g., anticalins) via SpyCatcher-SpyTag protein-peptide interactions.

[0306] Fig.44 AE. Enhancement of NTAA cleavage reaction using hybridization of cleavage agents with recording tags (A) and (B). The recording tag-labeled peptide attached to a solid phase matrix (e.g., beads) is modified or labeled on the NTAA (Mod), for example, with PITC, DNP, SNP, acetyl modifier, guanidation, etc. (C) A cleavage enzyme (e.g., acyl peptide hydrolase (APH), aminopeptidase (AP), Edmanase, etc.) is attached to a DNA tag that contains a universal primer sequence complementary to the universal primer sequence on the recording tag. The cleavage enzyme is recruited to the modified NTAA by hybridization of the DNA tag of the cleavage enzyme and the complementary universal primer sequence on the recording tag. (D) This hybridization step greatly increases the effective affinity of the cleavage enzyme for the NTAA. (E) The cleaved NTAA diffuses away and the associated cleavage enzyme can be removed by stripping off the hybridized DNA tag.

[0307] Fig.45 . Cyclic degradation peptide sequencing using peptide ligase + protease + diaminopeptidase. Butelase I connects the TEV-Butelase I peptide substrate (TENLYFQNHV, SEQ ID NO: 132) to the NTAA of the peptide to be tested. Butelase requires an NHV motif at the C-terminus of the peptide substrate. After ligation, the chimeric peptide substrate is cleaved after the glutamine (Q) residue using tobacco etch virus (TEV) protease, leaving a chimeric peptide with an asparagine (N) residue connected to the N-terminus of the peptide to be tested. Diaminopeptidase (DAP) or dipeptidyl peptidase, which cleaves two amino acid residues from the N-terminus, effectively removes the asparagine residue (N) and the original NTAA on the peptide to be tested by two amino acids to shorten the peptide to be tested added at the N-terminus. The newly exposed NTAA is read using the binder provided in this article, and the entire cycle is repeated "n" times for the "n" amino acids sequenced. The use of streptavidin-DAP metalloenzyme chimeric proteins and the tethering of a biotin group to the N-terminal asparagine residue allowed the control of DAP biosynthesis capacity. Detailed description

[0309] Terms not specifically defined herein should be given meanings by those skilled in the art based on the present disclosure and context. However, as used in the specification, unless otherwise specified, these terms have the indicated meanings.

[0310] I. Introduction

[0311] The present disclosure provides, in part, highly parallel, high-throughput methods for digital characterization and quantification of macromolecules, which have direct applications in the characterization and sequencing of proteins and peptides (see Figure 1B , Figure 2A). The method described herein uses a binding agent comprising a coding tag, the coding tag having identification information in the form of a nucleic acid molecule or a sequenceable polymer, wherein the binding agent interacts with the macromolecule of interest. A plurality of continuous binding cycles are performed, each cycle comprising a plurality of macromolecules fixed on a solid support, preferably representing a pooled sample, exposed to a plurality of binding agents. During each binding cycle, the identity of each binding agent binding the macromolecule is recorded by transferring the information from the binding agent coding tag to the recording tag co-located with the macromolecule, and the number of binding cycles can also be recorded. In an alternative embodiment, the information from the recording tag containing the identification information of the relevant macromolecule can be transferred to the coding tag (e.g., forming an extended coding tag) or the third "di-tag" construct of the bound binding agent. The binding events of multiple cycles construct historical binding information on the recording tag co-located with the macromolecule, thereby generating an extended recording tag comprising a plurality of coding tags, wherein the plurality of coding tags represent the temporary binding history of a given macromolecule in a colinear order. Additionally, a cycle specific encoded tag may be employed to track information from each cycle so that if a cycle is skipped for some reason, the extended record tag may continue to collect information in subsequent cycles and identify the cycle in which the information is missing.

[0312] Alternatively, information can be transferred from the record tag containing the identification information of the relevant macromolecule to the coding tag forming the extended coding tag or the third di-tag construct, to replace the information written or transferred to the record tag from the coding tag. The extended coding tag or di-tags formed can be collected after each binding cycle, which is convenient for subsequent sequence analysis. The identification information on the record tag containing a barcode (e.g., partition label, compartment label, sample label, graded object label, UMI or any combination thereof) can be used to map the extended coding label or di-tag sequence reading back to the original macromolecule. In this way, a nucleic acid encoding library representing the macromolecule binding history is generated. The nucleic acid encoding library can be amplified and analyzed using a high-throughput next-generation digital sequencing method, so that each run can analyze millions to billions of molecules. Creating a nucleic acid binding information encoding library is also useful on the other hand because it can be enriched, deducted and standardized by using a DNA-based technology that utilizes hybridization. These DNA-based methods are easy to quickly expand and customize, and are more cost-effective than methods that directly manipulate other types of macromolecular libraries (such as protein libraries). Therefore, nucleic acid-encoded binding information libraries can be processed by one or more techniques before sequencing to enrich and / or deduct and / or standardize the representation of the sequences. This makes it more efficient, faster, and more cost-effective to extract the most needed information from very large libraries, where individual members may initially vary by multiple orders of magnitude in abundance. Importantly, these nucleic acid-based techniques for manipulating library representation are orthogonal to more conventional methods and can be used in combination with them. For example, protein-based methods can be used to deduct common high-abundance proteins, such as albumin, to remove most but not all unwanted proteins. Subsequently, albumin-specific members of the extended record tag library can also be deducted, thereby achieving a more complete overall deduction.

[0313] On the one hand, the disclosure provides a highly parallelized method for peptide sequencing using an Edman-like degradation method, allowing sequencing from a large number of DNA record tag-labeled peptides (e.g., millions to billions). The peptides of these record tag labels are derived from proteolytic digestion or limited hydrolysis of protein samples, and the peptides of the record tag labels are randomly fixed on a sequencing matrix (e.g., porous beads) with appropriate intermolecular spacing. The modification of the N-terminal amino acid (NTAA) residue of the peptide with a small chemical group, such as phenylthiocarbamoyl (PTC), dinitrophenol (DNP), sulfonyl nitrophenol (SNP), dansyl, 7-methoxycoumarin, acetyl or guanidinyl, catalyzes or recruits NTAA cleavage reactions to allow cyclic control of the Edman-like degradation process. The modified chemical group can also provide enhanced binding affinity for homologous NTAA binding agents. By combining a homologous NTAA binding agent comprising a coding tag, and transferring the coding tag information (e.g., an encoder sequence providing identification information of the binding agent) from the coding tag to the recording tag (e.g., primer extension or connection), the modified NTAA of each immobilized peptide is identified. Subsequently, the modified NTAA is removed by chemical or enzymatic methods. In certain embodiments, an enzyme (e.g., Edmanase) is engineered to catalyze the removal of the modified NTAA. In other embodiments, naturally occurring exopeptidases, such as aminopeptidases or acyl peptide hydrolases, can be transformed to cut the terminal amino acids only under suitable chemical modification conditions.

[0314] II. Definitions

[0315] In the following description, certain specific details are set forth in order to provide a thorough understanding of various embodiments. However, those skilled in the art will appreciate that the compounds of the present invention can be prepared and used without these details. In other cases, well-known structures are not shown or described in detail to avoid unnecessarily obscuring the description of the embodiments. Unless the context otherwise requires, throughout the specification and the subsequent claims, the words "include" and their variants, such as "include" and "comprising", should be interpreted in an open, inclusive sense, i.e., "including but not limited to". In addition, the term "include" (and related terms such as "include" or "includes" or "have" or "includes") is not intended to exclude certain other embodiments, for example, embodiments of any composition. The substances, compositions, methods or processes described herein, etc., may be "composed of" or "consisting essentially of..." by the described features. The titles provided herein are for convenience only and do not explain the scope or meaning of the claimed embodiments.

[0316] Reference throughout this specification to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the phrases "in one embodiment" or "in an embodiment" appearing in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0317] As used herein, the singular forms "a," "an," and "the" include plural references unless otherwise specified. Thus, for example, reference to "a peptide" includes one or more peptides or mixtures of peptides. Furthermore, unless otherwise specified or obvious from the context, as used herein, the term "or" should be understood to be inclusive and encompass both "or" and "and."

[0318] As used herein, the term "macromolecule" includes large molecules composed of smaller subunits. Examples of macromolecules include, but are not limited to, peptides, polypeptides, proteins, nucleic acids, carbohydrates, lipids, macrocyclic compounds. Macromolecules also include chimeric macromolecules, which are composed of a combination of two or more types of macromolecules covalently linked together (e.g., peptides linked to nucleic acids). Macromolecules may also include "macromolecular assemblies," which are composed of non-covalent complexes of two or more macromolecules. Macromolecular assemblies can be composed of the same type of macromolecule (e.g., protein-protein) or two different types of macromolecules (e.g., protein-DNA).

[0319] As used herein, the term "peptide" includes peptides, polypeptides and proteins, and refers to a molecule comprising a chain of two or more amino acids connected by a peptide bond. In general, peptides having more than 20-30 amino acids are commonly referred to as polypeptides, while peptides having more than 50 amino acids are commonly referred to as proteins. The amino acids of the peptide are most typically L-amino acids, but may also be D-amino acids, modified amino acids, amino acid analogs, amino acid mimetics or any combination thereof. Peptides may be naturally occurring, synthetically produced or recombinantly expressed. Peptides may also include other groups that modify the amino acid chain, for example, functional groups added by post-translational modifications.

[0320] As used herein, the term "amino acid" refers to an organic compound containing an amine group, a carboxylic acid group, and a side chain specific to each amino acid, which serves as a monomer subunit of a peptide. Amino acids include the 20 standard, naturally occurring or typical amino acids as well as non-standard amino acids. Standard natural 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 modified amino acids, amino acid analogs, amino acid mimetics, non-standard proteinogenic amino acids, or naturally occurring or chemically synthesized non-proteinogenic amino acids. Examples of non-standard amino acids include, but are not limited to, selenocysteine, pyrrolysine and N-formylmethionine, β-amino acids, homologous amino acids, proline and pyruvate derivatives, 3-substituted alanine derivatives, glycine derivatives, ring-substituted phenylalanine and tyrosine derivatives, linear core amino acids, N-methyl amino acids.

[0321] As used herein, the term "post-translational modification" refers to modifications that occur on a peptide after the peptide has been translated by the ribosome. The post-translational modification can be a covalent modification or an enzymatic modification. Examples of post-translational modifications include, but are not limited to, acylation, acetylation, alkylation (including methylation), biotinylation, butyrylation, carbamylation, carbonylation, deamidation, deiminiation, dinaphthylamine formation, disulfide bridge formation, eliminylation, flavin attachment, formylation, γ-carboxylation, glutamylation, glycylation, glycosylation, glypiation, heme C attachment, hydroxylation, hypusine formation, iodination, prenylation, lipidation, lipoylation, propionylation, methylation, myristoylation, oxidation, palmitoylation, pegylation, phosphoesterification, phosphorylation, prenylation, propionylation, retinylidene Schiff base formation, S-glutathionylation, S-nitrosylation, S-sulfenylation, selenoylation, succinylation, sulfation, ubiquitination, and C-terminal amidation. Post-translational modifications include modifications of the amino terminus and / or carboxyl terminus of the 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 carboxyl group include, but are not limited to, amide, lower alkyl amide, dialkyl amide, and lower alkyl ester modifications (e.g., wherein the lower alkyl is C 1 -C 4 The term post-translational modification may also include modifications of the peptides that include one or more detectable labels, such as, but not limited to, modifications of the amino acids that fall between the amino and carboxyl termini as described above.

[0322] As used herein, the term "binding agent" refers to a nucleic acid molecule, peptide, polypeptide, protein, carbohydrate or small molecule that binds, associates, associates, recognizes or combines with a macromolecule or a component or feature of a macromolecule. The binding agent can form a covalent association or a non-covalent association with a macromolecule or a component or feature of a macromolecule. The binding agent can also be a chimeric binding agent, consisting of two or more types of molecules, such as a nucleic acid molecule-peptide chimeric binding agent or a carbohydrate-peptide chimeric binding agent. The binding agent can be a naturally occurring, synthetically produced or recombinantly expressed molecule. The 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 connected subunits of a macromolecule (e.g., a dipeptide, a tripeptide, a higher order peptide of a long peptide, a polypeptide or a protein molecule). The binding agent can bind to a linear molecule or a molecule with a three-dimensional structure (also called 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. The binding agent can bind to an N-terminal peptide, a C-terminal peptide, or an intervening peptide of a peptide, polypeptide or protein molecule. The binding agent can bind to the N-terminal amino acid, C-terminal amino acid or intervening amino acid of the peptide molecule. Compared to unmodified or unlabeled amino acids, the binding agent can preferentially bind to chemically modified or labeled amino acids. For example, the binding agent can preferably bind to amino acids modified with acetyl groups, amidino groups, dansyl groups, PTC groups, DNP groups, SNP parts, etc. instead of being bound to amino acids without these groups. The binding agent can be bound to the post-translational modification of the peptide molecule. The binding agent can show selective binding with components or features of macromolecules (for example, the binding agent can selectively bind to one of 20 possible natural amino acid residues and bind to other 19 natural amino acid residues with very low affinity or not at all). The binding agent can show lower selective binding, wherein the binding agent can bind to multiple components or features of macromolecules (for example, the binding agent can bind to two or more different amino acid residues with similar affinity). The binding agent comprises a coding tag, which is connected to the binding agent by a joint.

[0323] As used herein, the term "joint" refers to one or more of a nucleotide, nucleotide analog, amino acid, peptide, polypeptide or non-nucleotide chemical group used to connect two molecules. The joint can be used to combine a binding agent and a code tag, a record tag and a macromolecule (e.g., a peptide), a macromolecule and a solid support, etc., a record tag and a solid support. In certain embodiments, the joint connects two molecules by an enzymatic reaction or a chemical reaction (e.g., click chemistry).

[0324] As used herein, the term "proteomics" refers to the quantitative analysis of the proteome within cells, tissues and body fluids, and the analysis of the corresponding spatial distribution of the proteome within the cells and tissues. In addition, proteomic studies include the dynamic state of the proteome as a function of biological and defined biological or chemical stimuli that change over time.

[0325] As used herein, the term "non-cognate binder" refers to a "homologous binder" that cannot bind to a macromolecular feature, component, or subunit being measured with low affinity in a specific binding cycle reaction, while a "homologous binder" binds to the corresponding macromolecular feature, component, or subunit with high affinity. For example, if a tyrosine residue of a peptide molecule is measured in a binding reaction, non-cognate binders refer to those binders that bind to the tyrosine residue with low affinity or do not bind to the tyrosine residue at all, so that under conditions suitable for transferring the coded tag information from the homologous binder to the record tag, the non-cognate binder cannot transfer the coded tag information to the record tag. Alternatively, if a tyrosine residue of a peptide molecule is measured in a binding reaction, non-cognate binders are those agents that bind to the tyrosine residue with low affinity or do not bind to the record tag at all, so that under conditions suitable for embodiments involving extended coded tags rather than extended record tags, the record tag information cannot be effectively transferred to the coded tag.

[0326] The terminal amino acid at one end of the peptide chain with a free amino group is referred to herein as the "N-terminal amino acid" (NTAA). The terminal amino acid at the other end of the chain with a free carboxyl group is referred to herein as the "C-terminal amino acid" (CTAA). The amino acids that make up the peptide can be numbered in sequence, where the length of the peptide is "n" amino acids. As used herein, NTAA is considered to be the nth amino acid (also referred to herein as "nNTAA"). Using this nomenclature, the next amino acid is the n-1st amino acid, followed by the n-2nd amino acid, and so on for the peptide length from the N-terminus to the C-terminus. In certain embodiments, NTAA, CTAA, or both can be modified or labeled with the chemical groups.

[0327] As used herein, the term "barcode" refers to a nucleic acid molecule of about 2 to about 30 bases (e.g., 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 or 30 bases) that provides a unique identifier tag or origin information for a macromolecule (e.g., protein, polypeptide, peptide), a binding agent, a binding agent group from a binding cycle, a sample macromolecule, a sample group, a macromolecule in a compartment (e.g., a droplet, a bead, or an isolated position), a macromolecule set in a compartment, a macromolecule fractionation, a macromolecule fractionation set, a spatial region or a spatial region set, a macromolecule library, or a binding agent library. A barcode can be an artificial sequence or a naturally occurring sequence. In certain embodiments, each barcode in a barcode population is different. In other embodiments, a portion of the barcodes in the barcode population are different, for example, 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 in the barcode population are different. The barcode population can be generated randomly or non-randomly. In certain embodiments, the barcode population is an error correction barcode. Barcodes can be used to computationally deconvolve multiplexed sequencing data and identify sequence reads originating from a single macromolecule, sample, library, etc. Barcodes can also be used to deconvolute a collection of macromolecules that have been distributed into small compartments to enhance mapping. For example, instead of mapping a peptide back to the proteome, the peptide is mapped back to its origin protein molecule or protein complex.

[0328] A “sample barcode,” also called a “sample tag,” identifies which sample a macromolecule came from.

[0329] "Spatial barcodes" which are regions of 2-D or 3-D tissue sections derived from macromolecules. Spatial barcodes can be used for molecular pathology on tissue sections. Spatial barcodes allow multiplexed sequencing of multiple samples or libraries from tissue sections.

[0330] As used herein, the term "coding tag" refers to a nucleic acid molecule of about 2 bases to about 100 bases, including any integers including 2 and 100 and therebetween, which contains identification information of its associated binding agent. "Coding tags" can also be made of "sequenceable polymers" (see, e.g., Niu et al., 2013, Nat. Chem. 5: 282-292; Roy et al., 2015, Nat. Commun. 6: 7237; Lutz, 2015, Macromolecules 48: 4759-4767; each of which is incorporated by reference as a whole). The coding tag comprises an encoder sequence, which is optionally flanked by a spacer on one side or by a spacer on each side. The coding tag may also include an optional UMI and / or an optional binding cycle-specific barcode. The coding tag may be single-stranded or double-stranded. The double-stranded coding tag may include a blunt end, an overhanging end, or both. The coding tag may refer to a coding tag directly attached to a binding agent, a complementary sequence hybridized to a coding tag directly attached to a binding agent (e.g., for double-stranded coding tags), or the coding tag information in an extended record tag. In certain embodiments, the coding tag may further comprise a binding cycle-specific spacer or barcode, a unique molecular identifier, a universal priming site, or any combination thereof.

[0331] As used herein, the term "encoder sequence" or "encoder barcode" refers to a nucleic acid molecule of about 2 bases to about 30 bases (e.g., 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 or 30 bases) length, providing identification information of its associated binding agent. The encoder sequence can uniquely identify its associated binding agent. In certain embodiments, the encoder sequence provides identification information of its associated binding agent and the binding cycle using the binding agent. In other embodiments, the encoder sequence is combined with a separate binding cycle-specific barcode in the coding tag. Alternatively, the encoder sequence can identify its associated binding agent as a member of a group comprising two or more different binding agents. In some embodiments, this level of recognition is sufficient for analytical purposes. For example, in some embodiments involving binding agents that bind to amino acids, it may be sufficient to know that a peptide contains one of two possible amino acids at a specific position without explicitly identifying the amino acid residue at that position. In another example, a common coding sequence is used for a polyclonal antibody, which contains a mixture of antibodies that recognize more than one epitope of a protein target and has different specificities. In other embodiments, in the case where the encoder sequence recognizes a set of possible binding agents, a sequential decoding method can be used to generate a unique identification for each binding agent. This is achieved by changing the coding sequence of a given binding agent in repeated binding cycles (see Gunderson et al., 2004, Genome Res. 14: 870-7). When combined with the coding information from other cycles, the partial identification coding tag information from each binding cycle is a unique identifier generated for the binding agent, for example, a specific combination of coding tags rather than separate coding tags (or encoder sequences) is provided to generate the unique identification information for the binding agent. Preferably, the coding sequences within the binding agent library have the same or similar number of bases.

[0332] As used herein, the terms "binding cycle-specific tag," "binding cycle-specific barcode," or "binding cycle-specific sequence" refer to a unique sequence used to identify a library of binders used within a particular binding cycle. A binding cycle-specific tag may comprise a length of about 2 bases to about 8 bases (e.g., 2, 3, 4, 5, 6, 7, or 8 bases). A binding cycle-specific tag may be incorporated into the encoding tag of a binder as part of a spacer sequence, part of an encoder sequence, part of a UMI, or as a separate component within the encoding tag.

[0333] As used herein, the term "spacer" (Sp) refers to a nucleic acid molecule of about 1 base to about 20 bases (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 bases) in length, present at the end of a record tag or a coding tag. In certain embodiments, the spacer sequence is flanked by the coding sequence of the coding tag at one or both ends. After the binding agent is bound to the macromolecule, the complementary spacer sequences on the relevant coding tag and the record tag are annealed separately, allowing the binding information to be transferred to the record tag, coding tag or di-complementary spacer construct by primer extension reaction or connection. Sp' refers to a spacer sequence complementary to Sp. Preferably, the spacer sequences within the binding agent library have the same number of bases. A common (shared or identical) spacer region can be used in the binding agent library. The spacer sequence may have a "cycle-specific" sequence to track the binding agent used in a specific binding cycle. The spacer sequence (Sp) can be constant in all binding cycles, specific to a particular class of macromolecules, or specific to the number of binding cycles. Macromolecule class-specific spacers allow the coded tag information of the homologous binder present in the extended record tag in the completed binding / extension cycle to be annealed to the coded tag of another binder, which recognizes the macromolecule of the same class through the class-specific spacer in subsequent binding cycles. Only the sequential binding of the correct homologous pair can produce the spacer element of interaction and effective primer extension. The spacer sequence can contain a sufficient number of bases to anneal with the complementary spacer sequence in the record tag to trigger primer extension (also called polymerase extension) reaction, or provide a "splint" for the connection reaction, or mediate a "sticky end" connection reaction. The spacer sequence can contain fewer bases than the encoder sequence in the coding tag.

[0334] As used herein, the term "record tag" refers to a nucleic acid molecule or a sequenceable polymer molecule (see, e.g., Niu et al., 2013, Nat. Chem. 5: 282-292; Roy et al., 2015, Nat. Bulletin 6: 7237; Lutz, 2015, Macromolecules 48: 4759-4767; each of which is incorporated herein by reference in its entirety) that contains identification information of an associated macromolecule. In certain embodiments, after a binding agent binds to a macromolecule, information from a coding tag attached to the binding agent can be transferred to the record tag associated with the macromolecule while the binding agent is bound to the macromolecule. In other embodiments, after a binding agent binds to a macromolecule, information from a record tag associated with a macromolecule can be transferred to the coding tag attached to the binding agent while the binding agent is bound to the macromolecule. The recoded tag can be directly attached to the macromolecule, attached to the macromolecule through a multifunctional linker, or attached to the macromolecule by virtue of its proximity (or co-localization) on a solid support. The record tag can be connected through its 5' end or 3' end or internal site, as long as the link is compatible with the method for transferring the coded tag information to the record tag, and vice versa. The record tag can also include other functional components, such as a universal priming site, a unique molecular identifier, a barcode (e.g., a sample barcode, a graded barcode, a spatial barcode, a compartment label, etc.), a spacer sequence complementary to the spacer sequence of the coded tag, or any combination thereof. In an embodiment in which the coded tag information is transferred to the record tag using polymerase extension, the spacer sequence of the record tag is preferably located at the 3' end of the record tag.

[0335] As used herein, the term "primer extension", also referred to as "polymerase extension", refers to a reaction catalyzed by a nucleic acid polymerase (e.g., DNA polymerase) in which a nucleic acid molecule (e.g., oligonucleotide primer, spacer sequence) annealed to a complementary strand is extended by the polymerase using the complementary strand as a template.

[0336] As used herein, the term "unique molecular identifier" or "UMI" refers to a nucleic acid molecule of about 3 to about 40 bases As used herein, the term "unique molecular identifier" or "UMI" refers to a nucleic acid molecule of about 3 to about 40 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, or 40 bases) in length that provides a unique identifier tag for each macromolecule (e.g., peptide) or binder to which the UMI is attached. The macromolecule UMI can be used to computationally deconvolute sequencing data from multiple extended record tags to identify extended record tags originating from a single macromolecule. The binder UMI can be used to identify each individual binder that binds to a specific macromolecule. For example, a UMI can be used to identify the number of individual binding events of a binder specific for a single amino acid that occurs for a particular peptide molecule. It should be understood that when both a UMI and a barcode are referenced in the context of a binder or macromolecule, the barcode refers to identification information of the individual binder or macromolecule other than the UMI (e.g., sample barcode, compartment barcode, binding cycle barcode).

[0337] As used herein, the term "universal priming site" or "universal primer" or "universal priming sequence" refers to a nucleic acid molecule that can be used for library amplification and / or sequencing reactions. The universal priming site can include, but is not limited to, a priming site (primer sequence) for PCR amplification, a flow channel adapter sequence that anneals to a complementary oligonucleotide on the flow channel surface (which facilitates bridge amplification in some next-generation sequencing platforms), a sequencing priming site, or a combination thereof. The universal priming site can be used for other types of amplifications, including amplifications that are typically used in conjunction with next-generation digital sequencing. For example, extended record tag molecules can be circularized and the universal priming site is used for rolling circle amplification to form DNA nanoballs that can be used as sequencing templates (Drmanac et al., 2009, Science 327: 78-81). Alternatively, the record tag molecules can be directly circularized and sequenced by polymerase extension from the universal priming site (Korlach et al., 2008, Proc. Natl. Acad. Sci. 105: 1176-1181). The term "forward" when used in the context of a "universal priming site" or "universal primer" may also be referred to as "5'" or "sense". The term "reverse" when used in the context of a "universal priming site" or "universal primer" may also be referred to as "3'" or "antisense".

[0338] As used herein, the term "extended recording label" refers to a recording label to which the information of the coding label (or its complementary sequence) of at least one binding agent has been transferred after the binding agent is bound to the macromolecule. The information of the coding label can be transferred to the recording label directly (e.g., ligation) or indirectly (e.g., primer extension). The information of the coding label can be transferred to the recording label enzymatically or chemically. The extended recording label can include information of the coding label that can be transferred to the recording label enzymatically or chemically. The extended record tag may contain 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,45,50,55,60,65,70,75,80,85,90,95,100,125,150,175,200 or more binding agent information of the coding tag. The base sequence of the extended record tag may reflect the binding time and order of the binding agent identified by its coding tag. The partial binding order of the binding agent identified by the coding tag may be reflected, or the binding order of any binding agent identified by the coding tag may not be reflected. In certain embodiments, the encoded tag information present in the extended record tag represents the sequence of the macromolecule analyzed with at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity. In certain embodiments where the extended record tag does not represent the sequence of the macromolecule analyzed with 100% identity, the error may be due to off-target binding of the binding agent, or due to a "missed" binding cycle (e.g., because the binding agent cannot bind to the macromolecule during the binding cycle because the primer extension reaction fails), or both.

[0339] As used herein, the term "extended coding tag" refers to a coding tag to which the information of at least one recording tag (or its complementary sequence) is transferred after a binding agent that binds to the coding tag binds to a macromolecule, and the recording tag is associated with the macromolecule. The information of the recording tag can be transferred to the coding tag directly (e.g., connection) or indirectly (e.g., primer extension). The information of the recording tag can be transferred enzymatically or chemically. In certain embodiments, the extended coding tag contains the information of the recording tag, reflecting a binding event. As used herein, the term "di-tag" or "di-tag construct" refers to a nucleic acid molecule to which the information of at least one recording tag (or its complementary sequence) and at least one coding tag (or its complementary sequence) is transferred after binding to a binding agent connected to the coding tag, and the recording tag is associated with the macromolecule (see, Fig.11 B). The information of the recording tag and the coding tag can be transferred to the di-tag indirectly (e.g., primer extension). The information of the recording tag can be transferred enzymatically or chemically. In some embodiments, the di-tag comprises a UMI of the recording tag, a compartment label of the recording tag, a universal priming site of the recording tag, a UMI of the coding tag, a coding sequence of the coding tag, a combined cycle-specific barcode, a universal priming site of the coding tag, or any combination thereof.

[0340] As used herein, the terms "solid support," "solid surface," or "solid matrix" or "matrix" refer to any solid material, including porous and non-porous materials, to which macromolecules (e.g., peptides) can be directly or indirectly bound by means known in the art, including covalent and non-covalent interactions, or any combination thereof. A solid support can be two-dimensional (e.g., a planar surface) or three-dimensional (e.g., a gel matrix or beads). A solid support can be any support surface, including, but not limited to, beads, microbeads, arrays, glass surfaces, silicon surfaces, plastic surfaces, filters, membranes, nylon, silicon crystal chips, fluidic chips, flow channels, biochips including signal transduction electronic devices, channels, microtiter wells, ELISA plates, rotating interferometry disks, nitrocellulose membranes, nitrocellulose-based polymer surfaces, polymer matrices, nanoparticles, or microspheres. The material for solid carrier includes but is not limited to acrylamide, agarose, cellulose, nitrocellulose, glass, gold, quartz, polystyrene, polyethylene vinyl acetate, polypropylene, polymethacrylate, polyethylene, polyethylene oxide, polysilicate, polycarbonate, Teflon, fluorocarbon, nylon, silicone rubber, polyanhydride, polyglycolic acid, polylactic acid, polyorthoester, functionalized silane, polypropyl fumarate, collagen, glycosaminoglycan, polyamino acid, dextran or any combination thereof.Solid support also includes film, membrane, bottle, dish, fiber, braided fiber, shaped polymer, such as pipe, particle, bead, microsphere, microparticle or any combination thereof.For example, when solid surface is bead, the bead can include but is not limited to ceramic bead, polystyrene bead, polymer bead, methyl styrene bead, agarose bead, acrylamide bead, solid core bead, porous bead, paramagnetic bead, glass bead or controlled pore bead.Bead can be spherical or irregular shape. The size of the beads can be nanometers, such as 100 nanometers, millimeters, such as 1 millimeter. In certain embodiments, the size range of the beads is from about 0.2 microns to about 200 microns, or from about 0.5 microns to about 5 microns. In some embodiments, the beads can be about 1,1.5,2,2.5,2.8,3,3.5,4,4.5,5,5.5,6,6.5,7,7.5,8,8.5,9,9.5,10,10.5,15, or 20 μm in diameter. In certain embodiments, a "bead" solid support can refer to an individual bead or a plurality of beads.

[0341] As used herein, the term "nucleic acid molecule" or "polynucleotide" refers to a single-stranded or double-stranded polynucleotide containing deoxyribonucleotides or ribonucleotides linked by 3'-5' phosphodiester bonds, as well as polynucleotide analogs. Nucleic acid molecules include, but are not limited to DNA, RNA and cDNA. Polynucleotide analogs can have backbones other than the standard phosphodiester bonds found in natural polynucleotides, and optionally have modified sugar groups other than ribose or deoxyribose. Polynucleotide analogs contain bases that can form hydrogen bonds with standard polynucleotide bases by Watson-Crick base pairing, wherein the analog backbone presents the bases in a manner that allows hydrogen bonding in a sequence-specific manner between the oligonucleotide analog molecule and the bases in the standard polynucleotide. Examples of polynucleotide analogs include, but are not limited to, xenogeneic nucleic acids (XNA), bridged nucleic acids (BNA), glycol nucleic acids (GNA), peptide nucleic acids (PNAs), γPNA, morpholino polynucleotides, locked nucleic acids (LNAs), threose nucleic acids (TNA), 2'-O-methyl polynucleotides, 2'-O-alkylribosyl substituted polynucleotides, phosphorothioate polynucleotides, and borophosphate polynucleotides. Polynucleotide analogs can 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 hypoxanthine, nitroazoles, isoquinolone analogs, azole carboxamides, and aromatic triazole analogs, or base analogs with additional functionality, such as a biotin group for affinity binding.

[0342] As used herein, "nucleic acid sequencing" refers to determining the order of nucleotides in a nucleic acid molecule or a sample of nucleic acid molecules.

[0343] As used herein, "next generation sequencing" refers to a high-throughput sequencing method that allows parallel sequencing of millions to billions of molecules. The example of the next generation sequencing method includes sequencing by synthesis, connection sequencing, hybridization sequencing, polony sequencing, ion semiconductor sequencing and pyrophosphate sequencing. By connecting primers to a solid substrate and to the complementary sequence of nucleic acid molecules, nucleic acid molecules can be hybridized with the solid substrate by the primers, and then amplified by using polymerase to produce multiple copies in discrete areas on the solid substrate (these groupings are sometimes referred to as polymerase clones polymerase colonies or polonies). Therefore, in the sequencing process, the nucleotides at a specific position can be sequenced multiple times (for example, hundreds or thousands of times)-this coverage depth is referred to as "deep sequencing". Examples of high-throughput nucleic acid sequencing technologies include platforms provided by Illumina, BGI, Qiagen, Thermo-Fisher, and Roche, including parallel bead arrays, synthesis sequencing, ligation sequencing, capillary electrophoresis, electronic microchips, "biochips," "microarrays, parallel microchips, and single-molecule array formats as reviewed by Service (Science 311: 1544-1546, 2006).

[0344] As used herein, "single molecule sequencing" or "third generation sequencing" refers to a new generation sequencing method, in which the reading from the single molecule sequencing instrument is generated by sequencing a single DNA molecule. Unlike the next generation sequencing method that relies on amplification to clone many DNA molecules in parallel for sequencing in a phased approach, single molecule sequencing examines a single DNA molecule and does not require amplification or synchronization. Single molecule sequencing includes methods that require pausing the sequencing reaction after each base incorporation ("wash and scan" cycle) and methods that do not require stopping between reading steps. Examples of single molecule sequencing methods include single molecule real-time sequencing (Pacific Biosciences), nanopore-based sequencing (Oxford Nanopore), double-stranded interruption nanopore sequencing, and direct imaging DNA with advanced microscopes.

[0345] As used herein, "analyzing" the macromolecule means that all or part of the components of the macromolecule are quantified, characterized, distinguished or a combination thereof. For example, analyzing a peptide, polypeptide or protein includes determining all or part of the amino acid sequence (continuous or non-continuous) of the peptide. Analyzing a macromolecule also includes partial identification of the components of the macromolecule. For example, partially identifying the amino acids in the macromolecular protein sequence can identify the amino acids in the protein as belonging to a possible subset of amino acids. The analysis usually starts with analyzing the nth NTAA, and then proceeds to the next amino acid (i.e., n-1, n-2, n-3, etc.) of the peptide. This is achieved by cutting the nth NTAA, thereby converting the n-1 amino acid of the peptide into an N-terminal amino acid (referred to herein as "n-1NTAA"). Analyzing the peptide can also include determining the presence and frequency of post-translational modifications on the peptide, which may include or not include information about the sequence of post-translational modifications on the peptide. Analyzing a peptide can also include determining the presence and frequency of an epitope in the peptide, which may include or not include information about the order or position of the epitope in the peptide. Analyzing the peptides may include combining different types of analyses, such as obtaining epitope information, amino acid sequence information, post-translational modification information, or any combination thereof.

[0346] As used herein, the term "compartment" refers to a physical area or volume that separates or partitions a subset of macromolecules from a macromolecular sample. For example, a compartment can separate a single cell from other cells, or separate a portion of a sample proteome from the rest of the sample's proteome. A compartment can be an aqueous compartment (e.g., a microfluidic droplet), a solid compartment (e.g., a Picotiter well or microtiter well on a plate, a tube, a vial, a gel bead), or a separation area on a surface. A compartment can contain one or more beads to which macromolecules can be fixed.

[0347] As used herein, the term "compartment tag" or "compartment barcode" refers to a single-stranded or double-stranded nucleic acid molecule of about 4 bases to about 100 bases (including 4 bases, 100 bases and any integer therebetween) of identification information of a component (e.g., a proteome of a single cell) contained in one or more compartments (e.g., a microfluidic droplet). A compartment barcode identifies a subset of macromolecules in a sample, such as a subset of a protein sample, which has been separated from multiple (e.g., millions to billions) compartments into the same physical compartment or compartment group. Therefore, even after the components are brought together, the compartment tag can be used to distinguish between components from one or more compartments with the same compartment tag and components in another compartment with different compartment tags. By marking the protein and / or peptide in each compartment or in a group of two or more compartments with a unique compartment tag, peptides derived from the same protein, protein complex, or cells in a single compartment or compartment group can be identified. The compartment tag comprises a barcode and an optional universal primer, and the barcode is optionally flanked by a spacer sequence on one or both sides. The spacer sequence may be complementary to the spacer sequence of the record tag, so that the compartment tag information can be transferred to the record tag. The compartment tag may also include a universal priming site, a unique molecular identifier (for providing identification information of the peptide connected thereto), or both, particularly for embodiments in which the compartment tag includes a record tag for the peptide analysis method described below. The compartment tag may include a functional group for coupling a peptide (e.g., aldehyde, NHS, mTet, alkyne, etc.). Alternatively, the compartment tag may include a peptide that includes a recognition sequence for a protein ligase to allow the compartment tag to be connected to a target peptide. The compartment may include a single compartment tag, a plurality of identical compartment tags reserved for optional UMI sequences, or two or more different compartment tags. In certain embodiments, each compartment includes a unique compartment tag (one-to-one mapping). In other embodiments, multiple compartments from a larger compartment group include the same compartment tag (many-to-one mapping). The compartment tag may be attached to a solid support within a compartment (e.g., a bead) or to the surface of the compartment itself (e.g., the surface of a Picotiter well). Alternatively, the compartment tag may be free in the solution within the compartment.

[0348] As used herein, the term "partitioning" refers to the random assignment of unique barcodes to subpopulations of macromolecules from a population of macromolecules within a sample. In certain embodiments, partitioning can be achieved by assigning macromolecules to compartments. A partition can consist of macromolecules within a single compartment or macromolecules from multiple compartments in a group of compartments.

[0349] As used herein, "partition tag" or "partition barcode" refers to a single-stranded or double-stranded nucleic acid molecule comprising about 4 bases to about 100 bases (including 4 bases, 100 bases and any integer therebetween) that contains identification information for a partition. In certain embodiments, the partition tag for a macromolecule refers to the same compartment tag generated by dividing the macromolecule into compartments labeled with the same barcode.

[0350] As used herein, the term "fractionate" refers to a subset of macromolecules (e.g., proteins) in a sample that has been selected from the rest of the sample or organelle using a physical or chemical separation method, such as by size, hydrophobicity, isoelectric point, affinity, etc. Separation methods include HPLC separation, gel separation, affinity separation, cell fractionation, organelle fractionation, tissue fractionation, etc. Fluid fluidity, magnetism, electrical current, mass, density, and similar physical properties can also be used for separation.

[0351] As used herein, the term "fractionator barcode" refers to a single-stranded or double-stranded nucleic acid molecule comprising about 4 bases to about 100 bases (including 4 bases, 100 bases, and any integer therebetween) that contains identification information for the macromolecules in the fractionator.

[0352] III. Methods for analyzing macromolecules

[0353] The method described herein provides a highly parallelized method for macromolecular analysis. The highly multiplexed large analysis combined with detection is converted into a nucleic acid molecule library read by the next generation sequencing method. The method provided here is particularly useful for protein and peptide sequencing.

[0354] In a preferred embodiment, a protein sample is labeled with at least one nucleic acid record tag at the single molecule level, and the nucleic acid record tag includes a barcode (e.g., a sample barcode, a compartment barcode) and an optional unique molecular identifier. The protein sample undergoes proteolytic digestion to produce a group of record tag-labeled peptides (e.g., millions to billions). These record tag-labeled peptides are randomly collected and fixed on a solid support. The collected, fixed record tag-labeled peptides undergo multiple continuous binding cycles, each of which includes exposure to a plurality of binding agents (e.g., binding agents of all 20 naturally occurring amino acids) labeled with a coding tag containing an encoder sequence that identifies the relevant binding agent. During each binding cycle, information about the binding of the binding agent to the peptide is captured by transferring the coding tag information of the binding agent to the record tag (or transferring the record tag information to the coding tag or transferring the record tag information and the coding tag information to a separate di-tag construct). After the binding cycle is completed, a library of extended record tags (or extended encoding tags or di-tag constructs) representing the binding history of the peptides assayed is generated, which can be analyzed using very high throughput next generation digital sequencing methods. The use of nucleic acid barcodes in the record tags allows deconvolution of large amounts of peptide sequencing data, for example, to identify which sample, cell, subset of a proteome, or protein a peptide sequence originates from.

[0355] In one aspect, a method for analyzing a macromolecule is provided, comprising the following steps: (a) providing a macromolecule and an associated or co-localized record label attached to a solid support; (b) contacting the macromolecule with a first binding agent capable of binding to the macromolecule, wherein the first binding agent comprises a first coding label having identification information about the first binding agent; (c) transferring information of the first coding label to the record label to produce a first sequence-extended record label; (d) contacting the macromolecule with a second binding agent capable of binding to the macromolecule, wherein the second binding agent comprises a second coding label having identification information about the second binding agent; (e) transferring information of the second coding label to the first sequence-extended record label to produce a second sequence-extended record label; and (f) analyzing the second sequence-extended record label (see, e.g., Figure 2A -D)

[0356] In some embodiments, the contacting steps (b) and (d) are performed sequentially, for example, the first binding agent and the second binding agent are contacted with the macromolecule in separate binding cycle reactions. In other embodiments, the contacting steps (b) and (d) are performed simultaneously, for example, the first binding agent, the second binding agent, and optionally additional binding agents are included in a single binding cycle reaction. In a preferred embodiment, the contacting steps (b) and (d) include contacting the macromolecule with multiple binding agents.

[0357] In some embodiments, the method further includes the following steps between steps (e) and (f): (x) repeating steps (d) and (e) one or more times by replacing the second binding agent with a third (or higher order) binding agent capable of binding to the macromolecule, wherein the third (or higher order) binding agent comprises a third (or higher order) coding tag having identification information about the third (or higher order) binding agent; (y) transferring the information of the third (or higher order) coding tag to the second (or higher order) extended recording tag to produce a third (or higher order) extended recording tag; and (z) analyzing the third (or higher order) extended recording tag.

[0358] The third (or higher) binding agent can be contacted with the macromolecule in a separate binding cycle reaction from the first binding agent and the second binding agent. Alternatively, the third (or higher) binding agent can be contacted with the macromolecule together with the first binding agent and the second binding agent in a single binding cycle reaction.

[0359] In a second aspect, a method for analyzing a macromolecule is provided, comprising the following steps: (a) providing a macromolecule, an associated first recording label and an associated second recording label connected to a solid support; (b) contacting the macromolecule with a first binding agent capable of binding to the macromolecule, wherein the first binding agent comprises a first coding label having identification information about the first binding agent; (c) transferring information of the first coding label to the recording label to produce a first order extended recording label; (d) contacting the macromolecule with a second binding agent capable of binding to the macromolecule, wherein the second binding agent comprises a second coding label having identification information about the second binding agent; (e) transferring information of the second coding label to the second recording label to produce a second order extended recording label; and (f) analyzing the first and second extended recording labels.

[0360] In some embodiments, contacting steps (b) and (d) are performed sequentially, for example, the first binding agent and the second binding agent are contacted with the macromolecule in separate binding cycle reactions. In other embodiments, the contacting steps (b) and (d) are performed simultaneously, for example, the first binding agent, the second binding agent, and optionally additional binding agents are included in a single binding cycle reaction.

[0361] In some embodiments, step (a) further comprises providing a third (or higher order) record tag associated with the solid support. In further embodiments, the method further comprises the following steps between steps (e) and (f): (x) repeating steps (d) and (e) one or more times by replacing the second binding agent with a third (or higher order) binding agent capable of binding to the macromolecule, wherein the third (or higher order) binding agent comprises a third (or higher order) coding tag having identification information about the third (or higher order) binding agent; (y) transmitting the information of the third (or higher order) coding tag to the second (or higher order) extended record tag to generate a third (or higher order) extended record tag; and (z) analyzing the third (or higher order) extended record tag.

[0362] The third (or higher) binding agent can be contacted with the macromolecule in separate binding cycle reactions with the first binding agent and the second binding agent. Alternatively, the third (or higher) binding agent can be contacted with the macromolecule together with the first binding agent and the second binding agent in a single binding cycle reaction.

[0363] In some embodiments, the first encoding tag, the second encoding tag, and any higher order encoding tags comprise a binding cycle-specific spacer sequence.

[0364] In a third aspect, a method for analyzing macromolecules is provided, comprising the following steps: (a) providing a peptide, and an associated record tag attached to a solid support; (b) modifying the N-terminal amino acid (NTAA) of the peptide with a chemical group to produce a modified NTAA; (c) contacting the peptide with a first binding agent capable of binding to the modified NTAA, wherein the first binding agent comprises a first coding tag having identification information of the first binding agent; (d) transferring the information of the first coding tag to the record tag to produce an extended record tag; (e) analyzing the extended record tag (see, e.g. Figure 3 ).

[0365] In some embodiments, step (c) further comprises contacting the peptide with a second (or higher order) binding agent comprising a second (or higher order) coding tag, wherein the second (or higher order) coding tag has identification information of the second (or higher order) binding agent, wherein the second (or higher order) binding agent is capable of binding to a modified NTAA other than the modified NTAA of step (b). In a further embodiment, after the peptide is contacted with the first binding agent, the peptide is contacted with the second (or higher order) binding agent in sequence, for example, the first binding agent and the second (or higher order) binding agent are contacted with the peptide in separate binding cycle reactions. In other embodiments, contacting the peptide with the second (or higher order) binding agent and the peptide with the first binding agent occur simultaneously, for example, the first binding agent and the second (or higher order) binding agent are included in a single binding cycle reaction.

[0366] In certain embodiments, the chemical group is added to NTAA by a chemical reaction or an enzymatic reaction.

[0367] In some embodiments, the chemical group used to modify the NTAA is phenylthiocarbamoyl (PTC), dinitrophenyl (DNP), sulfonylnitrophenyl (SNP), dansyl; 7-methoxycoumarin; thioacyl; thioacetyl; acetyl; guanidinyl; or thiobenzyl.

[0368] Chemical groups can be added to the NTAA by chemical reagents. In certain embodiments, the chemical reagent used to modify NTAA with a PTC group is phenyl isothiocyanate or its derivatives; the chemical reagent used to modify NTAA with a DNP group is 2,4-dinitrobenzenesulfonic acid (DNBS) or an aryl halide such as 1-fluoro-2,4-dinitrobenzene (DNFB); the chemical reagent used to modify NTAA with a sulfonyloxynitrobenzene (SNP) group is 4-sulfonyl-2-nitrofluorobenzene (SNFB); the chemical reagent used to modify NTAA with a dansyl group is a sulfonyl chloride, such as dansyl chloride; The chemical reagent used to modify NTAA with a 7-methoxycoumarin group is 7-methoxycoumarin acetic acid (MCA); the chemical reagent used to modify NTAA with a thioacyl group is a thioacylating agent; the chemical reagent used to modify NTAA with a thioacetyl group is a thioacetylation agent; the chemical reagent used to modify NTAA with an acetyl group is an acetylation agent (such as acetic anhydride); the chemical reagent used to modify NTAA with a guanidine group (amidine group) is a guanylating agent, or the chemical reagent used to modify NTAA with a thiobenzyl group is a thiobenzylation agent.

[0369] The fourth aspect provided by the present disclosure is a method for analyzing peptides, comprising the following steps: (a) providing a peptide and a related record label connected to a solid support; (b) modifying the N-terminal amino acid (NTAA) of the peptide with a chemical group to produce a modified NTAA; (c) contacting the peptide with a first binding agent capable of binding to the modified NTAA, wherein the first binding agent comprises a first coding label having identification information about the first binding agent; (d) transferring the information of the first coding label to the record label to produce a first extended record label; (e) removing the modified NTAA to expose a new NTAA; (f) modifying the new NTAA of the peptide with a chemical group to produce a newly modified NTAA; (g) contacting the peptide with a second binding agent capable of binding to the newly modified NTAA, wherein the second binding agent comprises a second coding label having identification information about the second binding agent; h) transferring the information of the second coding label to the first extended record label to produce a second extended record label; and (i) analyzing the second extended record label.

[0370] In some embodiments, the contacting steps (c) and (g) are performed sequentially, for example, the first binding agent and the second binding agent are contacted with the peptide in separate binding cycle reactions.

[0371] In some embodiments, the method further includes the following steps between steps (h) and (i): (x) repeating steps (e), (f) and (g) one or more times by replacing the second binding agent with a third (or higher order) binding agent capable of binding to the modified NTAA, wherein the third (or higher order) binding agent comprises a third (or higher order) coding tag having identification information about the third (or higher order) binding agent; (y) transferring the information of the third (or higher order) coding tag to the second (or higher order) extended recording tag to produce a third (or higher order) extended recording tag; and (z) analyzing the third (or higher order) extended recording tag.

[0372] In certain embodiments, the chemical group is added to the NTAA by a chemical reaction or an enzymatic reaction.

[0373] In some embodiments, the chemical group used to modify the NTAA is phenylthiocarbamoyl (PTC), dinitrophenyl (DNP), sulfonylnitrophenyl (SNP), dansyl; 7-methoxycoumarin; thioacyl; thioacetyl; acetyl; guanidinyl; or thiobenzyl.

[0374] Chemical groups can be added to the NTAA by chemical reagents. In certain embodiments, the chemical reagent used to modify NTAA with a PTC group is phenyl isothiocyanate or its derivatives; the chemical reagent used to modify NTAA with a DNP group is 2,4-dinitrobenzenesulfonic acid (DNBS) or an aryl halide such as 1-fluoro-2,4-dinitrobenzene (DNFB); the chemical reagent used to modify NTAA with a sulfonyloxynitrobenzene (SNP) group is 4-sulfonyl-2-nitrofluorobenzene (SNFB); the chemical reagent used to modify NTAA with a dansyl group is a sulfonyl chloride, such as dansyl chloride; The chemical reagent used to modify NTAA with a 7-methoxycoumarin group is 7-methoxycoumarin acetic acid (MCA); the chemical reagent used to modify NTAA with a thioacyl group is a thioacylating agent; the chemical reagent used to modify NTAA with a thioacetyl group is a thioacetylation agent; the chemical reagent used to modify NTAA with an acetyl group is an acetylation agent (such as acetic anhydride); the chemical reagent used to modify NTAA with a guanidine group (amidine group) is a guanylating agent, or the chemical reagent used to modify NTAA with a thiobenzyl group is a thiobenzylation agent.

[0375] In a fifth aspect, a method for analyzing a peptide is provided, comprising the following steps: (a) providing a peptide and an associated record label attached to a solid support; (b) contacting the peptide with a first binding reagent capable of binding to the N-terminal amino acid (NTAA) of the peptide, wherein the first binding reagent comprises a first coding label having identification information of the first binding reagent; (c) transferring the information of the first coding label to the record label to produce an extended record label; and (d) analyzing the extended record label.

[0376] In some embodiments, step (b) further comprises contacting the peptide with a second (or higher order) binding agent comprising a second (or higher order) coding tag, wherein the second (or higher order) coding tag has identification information of the second (or higher order) binding agent, wherein the second (or higher order) binding agent is capable of binding to NTAA other than the NTAA of the peptide. In a further embodiment, after the peptide is contacted with the first binding agent, the peptide is contacted with the second (or higher order) binding agent in order, for example, the first binding agent and the second (or higher order) binding agent are contacted with the peptide in separate binding cycle reactions. In other embodiments, contacting the peptide with the second (or higher order) binding agent and the peptide with the first binding agent occur simultaneously, for example, the first binding agent and the second (or higher order) binding agent are included in a single binding cycle reaction.

[0377] In a sixth aspect, a method for analyzing a peptide is provided, comprising the following steps: (a) providing a peptide and an associated record tag attached to a solid support; (b) contacting the peptide with a first binding agent capable of binding to the N-terminal amino acid (NTAA) of the peptide, wherein the first binding agent comprises a first coding tag having identification information about the first binding agent; c) transferring the information of the first coding tag to the record tag to produce a first extended record tag; (d) removing the modified NTAA to expose a new NTAA; (e) contacting the peptide with a second binding agent capable of binding to the newly modified NTAA, wherein the second binding agent comprises a second coding tag having identification information about the second binding agent; (f) transferring the information of the second coding tag to the first extended record tag to produce a second extended record tag; and (g) analyzing the second extended record tag.

[0378] In some embodiments, the method further includes the following steps between steps (f) and (g): (x) repeating steps (d), (e) and (f) one or more times by replacing the second binding agent with a third (or higher order) binding agent capable of binding to the modified macromolecule, wherein the third (or higher order) binding agent comprises a third (or higher order) coding tag having identification information about the third (or higher order) binding agent; and (y) transferring the information of the third (or higher order) coding tag to the second (or higher order) extended recording tag to produce a third (or higher order) extended recording tag; and analyzing the third (or higher order) extended recording tag in step (g).

[0379] In some embodiments, the contacting steps (b) and (e) are performed sequentially, for example, the first binding agent and the second binding agent are contacted with the peptide in separate binding cycle reactions.

[0380] In any of the embodiments provided, the method comprises analyzing a plurality of macromolecules in parallel. In a preferred embodiment, the method comprises analyzing a plurality of peptides in parallel.

[0381] In any of the embodiments provided herein, the step of contacting the macromolecule (or peptide) with a binding agent comprises contacting the macromolecule (or peptide) with a plurality of binding agents.

[0382] In any of the embodiments provided, the macromolecule can be a protein, a polypeptide or a peptide. In a further embodiment, wherein the peptide is obtained by fragmenting a protein isolated from a biological sample.

[0383] In any of the embodiments provided, the macromolecule can be or comprise a carbohydrate, a lipid, a nucleic acid, or a macrocycle.

[0384] In any of the embodiments provided, the recording label is a DNA molecule, a DNA molecule with modified bases, an RNA molecule, a BNA molecule, an XNA molecule, an LNA molecule, a PNA molecule, a γPNA molecule (Dragulescu-Andrasi et al., 2006, J. Am. Chem. Soc. 128: 10258-10267), a GNA molecule or any combination thereof.

[0385] In any of the embodiments provided herein, the record label comprises a universal priming site. In further embodiments, the universal priming site comprises a primer site for amplification, sequencing, or a combination of both.

[0386] In any of the embodiments provided herein, the record tag comprises a unique molecular identifier, a compartment tag, a partition barcode, a sample barcode, a fractionator barcode, a spacer sequence, or any combination thereof.

[0387] In any of the embodiments provided herein, the record tag comprises a unique molecular identifier (UMI), an encoder sequence, a binding cycle specific sequence, a spacer sequence, or any combination thereof.

[0388] In any of the embodiments provided herein, the binding cycle-specific sequence in the encoding tag can be a binding cycle-specific spacer sequence.

[0389] In certain embodiments, the binding cycle-specific sequence is encoded as a barcode separate from the encoder sequence.

[0390] In other embodiments, the encoder sequence and binding cycle-specific sequence are listed in a single barcode that is unique to the binding agent and each binding cycle.

[0391] In some embodiments, the spacer sequence comprises a common binding cycle sequence that is shared between binding agents from multiple binding cycles. In other embodiments, the spacer sequence comprises a unique binding cycle sequence that is shared between binding agents from the same binding cycle.

[0392] In any of the embodiments provided herein, the record label may comprise a bar code.

[0393] In any of the embodiments provided herein, the macromolecule and associated record label may be covalently attached to the solid support.

[0394] In any of the embodiments provided herein, the solid support is a bead, a porous bead, a porous matrix, an array, a glass surface, a silicon surface, a plastic surface, a filter, a membrane, nylon, a silicon chip, a flow chip, a biochip including a signal transduction electronic device, a microtiter well, an ELISA plate, a spinning interferometry disk, a nitrocellulose membrane, a nitrocellulose-based polymer surface, a nanoparticle or a microsphere.

[0395] In any of the embodiments provided herein, the solid support is a polystyrene bead, a polymer bead, agarose bead, acrylamide bead, solid core bead, porous bead, paramagnetic bead, glass bead or controlled pore bead.

[0396] In any of the embodiments provided herein, a plurality of macromolecules and associated record labels can be bound to the solid support. In further embodiments the plurality of macromolecules are spaced apart on the solid support at an average distance of ≥50 nm, ≥100 nm, or ≥200 nm.

[0397] In any of the embodiments provided herein, the binding agent can be a polypeptide or a protein. In further embodiments, the binding agent is a modified or variant aminopeptidase, a modified or variant aminoacyl-tRNA synthetase, a modified or variant anticalin, or a modified or variant ClpS.

[0398] In any of the embodiments provided herein, the binding agent can selectively bind to the macromolecule.

[0399] In any of the embodiments provided herein, the coding tag can be a DNA molecule, a DNA molecule with modified bases, an RNA molecule, a BNA molecule, an XNA molecule, an LNA molecule, a GNA molecule, a PNA molecule, a γPNA molecule, or a combination thereof.

[0400] In any of the embodiments provided herein, the binding agent and the coding tag can be connected by a linker.

[0401] In any of the embodiments provided herein, the binding agent and the coding tag are linked by a SpyTag / SpyCatcher or SnoopTag / SnoopCatcher peptide-protein pair (Zakeri, et al., 2012, Proc Natl Acad Sci USA 109(12):E690-697; Veggiani et al., 2016, Proc. Natl. Acad. Sci. USA 113:1202-1207, each of which is incorporated herein by reference in its entirety).

[0402] In any of the embodiments provided herein, the information transfer from the recording tag to the coding tag is mediated by DNA ligase. Alternatively, the information transfer from the recording tag to the coding tag is mediated by DNA synthesizer or chemical ligation.

[0403] In any of the embodiments provided herein, analyzing the extended record tag may include nucleic acid sequencing. In further embodiments, the nucleic acid sequencing method is synthetic sequencing, ligation sequencing, hybridization sequencing, polony sequencing, ion semiconductor sequencing or pyrophosphate sequencing. In other embodiments, the nucleic acid sequencing method is single molecule real-time sequencing, based on nanopore sequencing, or direct imaging of DNA using an advanced microscope.

[0404] In any of the embodiments provided herein, the extended record tags are amplified prior to analysis.

[0405] In any of the embodiments provided herein, the sequence of the encoded tag information contained in the extended record tags can provide information about the order of binding by the binding agent to the macromolecule and the sequence information of the analyte detected by the binding agent.

[0406] In any of the embodiments provided herein, the frequency of specific encoded tag information contained in the extended record tag (e.g., encoder sequence) can provide information about the frequency of binding by a specific binding agent to the macromolecule and the frequency of the analyte in the macromolecule detected by the binding agent.

[0407] In any embodiment disclosed herein, a plurality of macromolecular (e.g., protein) samples can be pooled, wherein the macromolecular population in each sample is labeled with a record tag comprising a sample-specific barcode. Such pooled macromolecular samples can be subjected to binding cycles in a single reaction tube.

[0408] In any of the embodiments disclosed herein, multiple extended record tags representing multiple macromolecules may be analyzed in parallel.

[0409] In any of the embodiments disclosed herein, multiple extended record tags representing multiple macromolecules can be analyzed in a multiplexed assay.

[0410] In any of the embodiments provided herein, the plurality of extended record tags are subjected to target abundance detection prior to analysis.

[0411] In any of the embodiments provided herein, the plurality of extended record tags are subjected to subtraction testing prior to analysis.

[0412] In any of the embodiments provided herein, the plurality of extended record tags are normalized prior to analysis to reduce high abundance species.

[0413] In any of the embodiments provided herein, the NTAA is removed using a modified aminopeptidase, a modified amino acid tRNA synthetase, mild Edman degradation, Edmanase, or anhydrous TFA.

[0414] In any of the embodiments provided herein, wherein at least one binding agent binds to a terminal amino acid residue. In some embodiments, the terminal amino acid residue is an N-terminal amino acid or a C-terminal amino acid.

[0415] In any of the embodiments provided herein, at least one of the binding agents binds to a post-translationally modified amino acid.

[0416] Features of the foregoing embodiments are provided in further detail in the following sections.

[0417] IV Macromolecules

[0418] In one aspect, a method for analyzing a macromolecule is provided, comprising the following steps: The macromolecule is a macromolecule composed of smaller subunits. In certain embodiments, the macromolecule is a protein, a protein complex, a polypeptide, a peptide, a nucleic acid molecule, a carbohydrate, a lipid, a macrocycle, or a chimeric macromolecule.

[0419] The macromolecules (e.g., proteins, polypeptides, peptides) analyzed according to the methods disclosed herein can be obtained from a suitable source or sample, including, but not limited to: biological samples, such as cells (primary cells and cultured cell lines), cell lysates or extracts, organelles or vesicles, including exosomes, tissues and tissue extracts; biopsies; stool; body fluids (such as blood, whole blood, serum, plasma, urine, lymph, bile, cerebrospinal fluid, interstitial fluid, aqueous or vitreous fluid, colostrum, sputum, amniotic fluid, saliva, anal and vaginal secretions, sweat and semen, exudates, exudates (e.g., fluid obtained from an abscess or any other site of infection or inflammation); Any organism, including samples of mammals, including samples containing microorganisms, which are preferred, and human samples, particularly preferably including samples containing microorganisms; environmental samples (such as air, agricultural, water and soil samples); microbial samples including samples from microbial biofilms and / or communities, and microbial spores; research samples, including extracellular fluid, extracellular supernatant from cell culture, inclusion bodies in bacteria, cellular compartments including mitochondrial compartments and periplasm.

[0420] In certain embodiments, the macromolecule is a protein, a protein complex, a polypeptide or a peptide. The amino acid sequence information and post-translational modification of the peptide, polypeptide or protein are transduced into a library encoded by nucleic acid, which can be analyzed by next generation sequencing methods. The peptide may contain L-amino acids, D-amino acids or both. The peptide, polypeptide, protein or protein complex may contain standard naturally occurring amino acids, modified amino acids (e.g., post-translational modifications), amino acid analogs, amino acid mimetics or any combination thereof. In some embodiments, the peptide, polypeptide or protein is naturally occurring, synthetically produced or recombinantly expressed. In any of the above peptide embodiments, the peptide, polypeptide, protein or protein complex may further contain post-translational modifications.

[0421] The standard natural 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). Nonstandard amino acids include selenocysteine, pyrrolysine and N-formylmethionine, β-amino acids, homologous 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.

[0422] Post-translational modifications (PTMs) of peptides, polypeptides or proteins 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, deamidation, dinaphthylamine formation, disulfide bridge formation, elimination, flavin attachment, formylation, γ-carboxylation, glutamylation, glycylation, glycosylation (e.g., N-linked, O-linked, C-linked, phosphoglycosylation), glypiation, heme C-linkage, hydroxylation, hypusine formation, iodination, prenylation, lipidation, lipidation, propionylation, methylation, myristoylation, oxidation, palmitoylation, pegylation, phosphoesterification, phosphorylation, prenylation, propionylation, retinyl Schiff base formation, S-glutathionylation, S-nitrosylation, S-sulfenylation, selenoylation, succinylation, sulfation, ubiquitination, and C-terminal amidation. Post-translational modification includes modification of the amino terminal and / or carboxyl terminal of a peptide, polypeptide or protein. Modification of the terminal amino group includes, but is not limited to, deamination, N-low alkyl, N-di-low alkyl and N-acyl modifications. Modification of the terminal carboxyl group includes, but is not limited to, amide, low alkyl amide, dialkyl amide and low alkyl ester modification (e.g., wherein the low alkyl is C1-C4 alkyl). Post-translational modification also includes modification of the amino acid falling between the amino and carboxyl terminal of a peptide, polypeptide or protein, such as, but not limited to, the above-mentioned modification. Post-translational modification can regulate the "biology" of intracellular proteins, such as its activity, structure, stability or positioning. Phosphorylation is the most common post-translational modification and plays an important role in the regulation of proteins, particularly in cell signaling (Prabakaran et al., 2012, Wiley Interdiscip Rev Syst Biol Med 4: 565-583). Adding sugars to proteins, such as glycosylation, has been shown to promote protein folding, improve stability and change regulatory functions. The attachment of lipids to proteins enables targeting of cell membranes. Post-translational modifications may also include modifications of the peptide, polypeptide or protein to include one or more detectable labels.

[0423] In certain embodiments, peptide, polypeptide or protein can be fragmented.For example, fragmented peptide can be obtained by separating protein from sample (for example biological sample).Peptide, polypeptide or protein can be fragmented by any method known in the art, including the fragmentation by protease or endopeptidase.In some embodiments, by using specific protease or endopeptidase targeting peptide, polypeptide or protein fragmentation.Specific protease or endopeptidase bind and cut at specific consensus sequence (for example, TEV protease specific to ENLYFQ\S consensus sequence).In other embodiments, the fragmentation of peptide, polypeptide or protein is non-targeted or random, by using non-specific protease or endopeptidase.Non-specific protease can bind and cut at specific amino acid residues instead of consensus sequence (for example, proteinase K is non-specific serine protease). Proteases and endopeptidases are well known in the art, and examples of proteases that can be used to cleave proteins or polypeptides into smaller peptide fragments include proteinase K, trypsin, chymotrypsin, pepsin, thermolysin, thrombin, factor Xa, furin, endopeptidase, papain, pepsin, subtilisin, elastase, enterokinase, Genenase, and mitogen-activated protein kinase. TM I, endoproteinase LysC, endoproteinase AspN, endoproteinase GluC, etc. (Granvogl et al., 2007, Anal Bioanal Chem 389: 991-1002). In certain embodiments, peptides, polypeptides or proteins are fragmented by proteinase K or an optional heat-labile form of proteinase K to enable rapid inactivation. Proteinase K is very stable in denaturing agents (such as urea and SDS) and can digest fully denatured proteins. Protein and polypeptide fragmentation into peptides can be performed before or after attaching a DNA tag or a DNA recording tag.

[0424] Chemical agents can also be used to digest proteins into peptide fragments. Chemical agents can cleave at specific amino acid residues (e.g., cyanogen bromide hydrolyzes the peptide bond at the C-terminus of a methionine residue). Chemical agents used to fragment polypeptides or proteins into smaller peptides include cyanogen bromide (CNBr), hydroxylamine, hydrazine, formic acid, BNPS-skatole [2-(2-nitrophenylsulfinyl)-3-methylindole], iodobenzoic acid, NTCB + Ni (2)-nitro-5-thiocyanatobenzoic acid) and the like.

[0425] In certain embodiments, after enzymatic or chemical cleavage, the resulting peptide fragments have approximately the same desired length, e.g., about 10 amino acids to about 70 amino acids, about 10 amino acids to about 60 amino acids, about 10 amino acids. About 50 amino acids, about 10 to about 40 amino acids, about 10 to about 30 amino acids, about 20 amino acids to about 70 amino acids, about 20 amino acids to about 60 amino acids, about 20 amino acids to about 50 amino acids, about 20 to about 40 amino acids, about 20 to about 30 amino acids, about 30 amino acids to about 70 amino acids, about 30 amino acids to about 60 amino acids, about 30 amino acids to about 50 amino acids, or about 30 amino acids to about 40 amino acids. By spiking a protein or polypeptide sample with a short test FRET (fluorescence resonance energy transfer) peptide comprising a peptide sequence containing a protease or endopeptidase cleavage site, the cleavage reaction can be preferably monitored in real time. In the complete FRET peptide, the fluorescent group and the quencher group are attached to either end of the peptide sequence containing the cleavage site, and the fluorescence resonance energy transfer between the quencher and the fluorophore results in low fluorescence. After the test peptide is cleaved with a protease or endopeptidase, the quencher and the fluorophore are separated, resulting in a great increase in fluorescence. When a certain fluorescence intensity is reached, the cleavage reaction can be stopped, thereby achieving a reproducible cleavage endpoint.

[0426] Macromolecular samples (e.g., peptides, polypeptides or proteins) can be subjected to protein fractionation methods before being attached to a solid support, wherein proteins or peptides are separated by one or more properties, such as cellular localization, molecular weight, hydrophobicity or isoelectric point or protein enrichment methods. Alternatively or in addition, protein enrichment methods can be used to select specific proteins or peptides (see, e.g., Whiteaker et al., 2007, Anal. Biochem. 362: 44-54, incorporated by reference as a whole) or select specific post-translational modifications (see, e.g., Huang et al., 2014. J. Chromatogr. A 1372: 1-17, incorporated by reference as a whole). Alternatively, proteins of a specific class or class such as immunoglobulins or immunoglobulin (Ig) isotypes such as IgG can be enriched or selected for analysis. In the case of immunoglobulin molecules, the analysis of sequences and abundance or frequency of hypervariable sequences involved in affinity binding is particularly interesting, particularly because their changes vary with disease progression or are associated with health, immunity and / or disease phenotypes. Overabundant proteins can also be subtracted from samples using standard immunoaffinity methods. Depletion of abundant proteins can be used for plasma samples, in which more than 80% of the protein components are albumin and immunoglobulins. There are several commercial products available for depletion of overabundant plasma samples, such as PROTIA and PROT20 (Sigma-Aldrich).

[0427] In certain embodiments, the macromolecule is composed of a protein or polypeptide. In one embodiment, the protein or polypeptide is labeled with a DNA record tag by standard amine coupling chemistry (see, e.g., Figure 2B , 2C, 28, 29, 31, 40). The -amino-amino groups (e.g., lysine residues) and the N-terminal amino group are particularly amenable to labeling with amine-reactive coupling agents, depending on the pH of the reaction (Mendoza and Vachet 2009). In certain embodiments (see, e.g., Figure 2B and Fig.29 ), the record tag comprises a reactive group (e.g., for conjugation to a solid surface, a multifunctional linker, or a macromolecule), a linker, a universal primer sequence, a barcode (e.g., a compartment tag, a partition barcode, a sample barcode, a graded barcode, or any combination thereof), an optional UMI, and a spacer (Sp) sequence for facilitating information transfer to / from the encoding tag. In another embodiment, the protein may be first labeled with a universal DNA tag, and the barcode-Sp sequence (representing a sample, a compartment, a physical location on a slide, etc.) is then attached to the protein by an enzymatic or chemical coupling step. (See, e.g., Figures 20, 30, 31, 40). The universal DNA tag comprises a short nucleotide sequence for labeling a protein or polypeptide macromolecule, which can also be used as an attachment point for a barcode (e.g., a compartment tag, a record tag, etc.). For example, the record tag may comprise a sequence complementary to the universal DNA tag at its end. In certain embodiments, the universal DNA tag is a universal primer sequence. After the universal DNA tag on the marker protein is hybridized with a complementary sequence in a record tag (e.g., bound to a bead), the annealed universal DNA tag can be amplified by primer extension to transfer the record tag information to the DNA-tagged protein. In a specific embodiment, the protein is labeled with a universal DNA tag before protease digestion into peptides. The universal DNA tag on the labeled peptide from the digest can then be converted into an information-rich and effective record tag.

[0428] In certain embodiments, the protein macromolecule can be immobilized to a solid support via an affinity capture reagent (and optionally covalently cross-linked), wherein the record tag is directly bound to the affinity capture reagent, or the protein can be directly immobilized to the solid support with the record tag (see, e.g. Figure 2C ).

[0429] V. Solid Support

[0430] The macromolecule of the present disclosure is connected to the surface of the solid support (also referred to as "matrix surface"). The solid carrier can be any porous or non-porous carrier surface, including but not limited to beads, microbeads, arrays, glass surfaces, silicon surfaces, plastic surfaces, filters, membranes, nylons, silicon crystal chips, flow channels, fluidic chips, biochips including signal transduction electronic devices, microtiter wells, ELISA plates, rotating interferometry disks, nitrocellulose membranes, polymer surfaces based on nitrocellulose, nanoparticles or microspheres. The materials used for solid carriers include but are not limited to acrylamide, agarose, cellulose, nitrocellulose, glass, gold, quartz, polystyrene, polyethylene vinyl acetate, polypropylene, polymethacrylate, polyethylene, polyethylene oxide, polysilicates, polycarbonates, Teflon, fluorocarbons, nylons, silicone rubbers, polyanhydrides, polyglycolic acid, polylactic acid, polyorthoesters, functionalized silanes, polypropyl fumarate, collagen, glycosaminoglycans, polyamino acids, dextran or any combination thereof. Solid supports also include films, membranes, bottles, plates, fibers, woven fibers, formed polymers, such as tubes, particles, beads, microspheres, microparticles, or any combination thereof. For example, when the solid surface is a bead, the bead may 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.

[0431] In certain embodiments, the solid support is a flow cell. The flow cell configuration can vary between different next-generation sequencing platforms. For example, the Illumina flow cell is a planar optically transparent surface similar to a microscope slide, which contains an oligonucleotide anchor lawn bound on its surface. The template DNA includes an adaptor connected to the end, which is complementary to the oligonucleotide on the flow cell surface. The adapted single-stranded DNA is combined with the flow cell and amplified by a "bridge" pattern solid phase PCR before sequencing. The 454 flow cell (54 Life Sciences) supports a "picotiter" plate, and the fiber optic slide has ~ 1.6 million 75Picotiter holes. Each single molecule of the sheared template DNA is captured on a separated bead, and each bead is compartmentalized in a dedicated droplet of an aqueous PCR reaction mixture in an oil emulsion. The template is cloned and amplified on the bead surface by PCR, and then the beads carrying the template are distributed to the wells of the Picotiter titration plate for sequencing reactions, and it is ideal that each well contains one or less beads. SOLiD (Supported Oligonucleotide Ligation and Detection) instruments from Applied Biosystems, such as the 454 system, amplify template molecules by emulsion PCR. After a step to remove beads that do not contain amplified template, the beads are stored in the flow cell. The flow cell can also be a simple filter glass, such as TWIST TMDNA synthesis columns (Glen Research).

[0432] In certain embodiments, solid support is a bead, which may refer to a single bead or a plurality of beads. In some embodiments, the bead is compatible with the next generation sequencing platform selected for downstream analysis (e.g., SOLiD or 454). In some embodiments, solid support is agarose beads, paramagnetic beads, polystyrene beads, polymer beads, acrylamide beads, solid beads, porous beads, paramagnetic beads, glass beads or controlled pore beads. In further embodiments, beads can be coated with binding functional groups (e.g., amine groups, streptavidin for binding biotin-labeled macromolecules, antibodies) to promote the combination with macromolecules.

[0433] The protein, polypeptide or peptide can be expressed by any means known in the art, including covalent and non-covalent interactions, or any combination thereof (see, e.g., Chan et al., 2007, PLoS One 2:e1164; Cazalis et al., Bioconj. Chem. 15:1005-1009; Soellner et al., 2003, J. Am. Chem. Soc. 125:11790-11791; Sun et al., 2006, Bioconjug. Chem. 17-52-57; Decreau et al., 2007, J. Org. Chem. 72:2794-2802; Camarero et al., 2004, J. Am. Chem. Soc. 126:14730-14731; Girish et al., 2003, J. Am. Chem. Soc. 127:154-155; al., 2005, Bioorg. Med. Chem. Lett. 15: 2447-2451; Kalia et al., 2007, Bioconjug. Chem. 18: 1064-1069; Watzke et al., 2006, Angew Chem. Int. Ed. Engl. 45: 1408-1412; Parthasarathy et al., 2007, Bioconjugate Chem. 18: 469-476; and Bioconjugate Techniques, GT Hermanson, Academic Press (2013), and each is incorporated herein by reference in its entirety) directly or indirectly attached to the solid support. For example, the peptide can be attached to the solid support by a ligation reaction. Alternatively, the solid support can include an agent or coating to facilitate direct or indirect attachment of the peptide to the solid support. Any suitable molecule or material can be used for this purpose, including proteins, nucleic acids, carbohydrates, and small molecules. For example, in one embodiment, the agent is an affinity molecule. In another example, the agent is an azide group that can react with an alkyne group in another molecule to promote the association or binding between the solid support and the other molecule.

[0434] Proteins, polypeptides or peptides can be attached to a solid support by a process known as "click chemistry". For this purpose, any rapid and essentially irreversible reaction can be used to attach a protein, polypeptide or peptide to the solid support. Exemplary reactions include the copper-catalyzed reaction of azides and alkynes to form triazoles (Huisgen 1,3-dipolar cycloaddition), strain-promoted azide alkyne cycloaddition (SPAAC), reactions of dienes and dienophiles (Diels-Alder), strain-promoted alkyne-nitrone cycloadditions, reactions of strained alkenes with azides, tetrazines or tetrazoles, [3+2] cycloadditions of alkenes and azides, Diels-Alder (IEDDA) reactions of alkenes and tetrazines with inverted electron requirements (e.g., m-tetrazine (mTet) and trans-cyclooctene (TCO)), photoreactions of alkenes and tetrazoles, Staudinger ligation of azides and phosphines, and various displacement reactions: for example, leaving group displacement by nucleophilic attack on an electrophilic atom (Horisawa 2014, Knall, Hollauf et al. 2014). Exemplary displacement reactions include the reaction of amines with: activated esters; N-hydroxysuccinimide esters; isocyanates; isothiocyanates, and the like.

[0435] In some embodiments, the macromolecule and solid support are connected by a functional group that can be formed by the reaction of two complementary reactive groups, such as a product of the aforementioned "click" reaction, a functional group. In various embodiments, the functional group can be formed by the reaction of an aldehyde, an oxime, a hydrazone, a hydrazide, an alkyne, an amine, an azide, an acyl azide, an acyl halide, a nitrile, a nitrone, a sulfhydryl, a disulfide, a sulfonyl halide, an isothiocyanate, an amide ester, an activated ester (e.g., N-hydroxysuccinimide ester, pentynoic acid STP ester), a ketone, an unsaturated carbonyl, an alkene, a maleimide, a haloimide, an epoxide, an aziridine, a tetrazine, a tetrazole, a phosphine, a biotin or an episulfide functional group with a complementary reactive group. Exemplary reactions are the reaction of an amine (e.g., a primary amine) with an N-hydroxysuccinimide ester or an isothiocyanate.

[0436] In other embodiments, the functional group comprises an alkene, an ester, an amide, a thioester, a disulfide, a carbocycle, a heterocycle or a heteroaryl. In further embodiments, the functional group comprises an alkene, an ester, an amide, a thioester, a disulfide, a carbocycle, a heterocycle or a heteroaryl. In other embodiments, the functional group comprises an amide or a thiourea. In some more specific embodiments, the functional group is a triazolyl functional group, an amide or a thiourea functional group.

[0437] In a preferred embodiment, iEDDA click chemistry is used to immobilize macromolecules (e.g., proteins, polypeptides, peptides) to solid supports because it is rapid and has high yields at low input concentrations. In another preferred embodiment, m-tetrazine is used instead of tetrazine in the iEDDA click chemistry reaction because m-tetrazine has better bond stability.

[0438] In a preferred embodiment, the substrate surface is functionalized with TCO and the recording label-labeled protein, polypeptide, peptide is immobilized on the TCO-coated substrate surface via the attached m-tetrazine moiety ( Fig.34 ).

[0439] Proteins, polypeptides or peptides can be fixed to the surface of a solid support through their C-terminus, N-terminus or internal amino acids, for example, through amines, carboxyls or sulfhydryls. Standard activated carriers for coupling amine groups include CNBr activated, NHS activated, aldehyde activated, azlactone activated and CDI activated carriers. Standard activated carriers for carboxyl coupling include carbodiimide activated carboxyl groups coupled to amine carriers. Cysteine ​​coupling can use maleimide, isoacetyl and pyridyl disulfide activated carriers. Another way to fix the carboxyl terminal of a peptide uses dehydrated trypsin, a catalytically inert derivative of trypsin, which binds to peptides containing lysine or arginine residues at the C-terminus without cleaving them.

[0440] In certain embodiments, a protein, polypeptide or peptide is immobilized to a solid support via covalent linkage of a solid surface-attached linker to a lysine group of the protein, polypeptide or peptide.

[0441] The recording label can be attached to the protein, polypeptide or peptide before or after being fixed to the solid support. For example, the protein, polypeptide or peptide can be first labeled with the recording label and then fixed to the solid surface via the recording label comprising two functional groups for coupling (see Fig.28 ). One functional group of the recording tag is coupled to a protein, while another functional group fixes the protein labeled by the recording tag on a solid support.

[0442] Alternatively, before the albumen, polypeptide or peptide are marked with the record label, the albumen, polypeptide or peptide are fixed to the solid support. For example, albumen can first be derivatized with a reactive group such as a click chemistry group. The activated protein molecule can then be connected to the suitable solid support, and then the record label is marked by adopting the complementary click chemistry group. As an example, the protein derived with alkynes and mTet groups can be fixed on the beads derived with azide and TCO, and attached to the record label marked with azide and TCO.

[0443] It should be understood that the methods provided herein for connecting a macromolecule (e.g., a protein, polypeptide, or peptide) to the solid support can also be used to attach a recording label to the solid support or to attach a recording label to a macromolecule (e.g., a protein, polypeptide, or peptide).

[0444] In certain embodiments, the surface of the solid support is passivated (blocked) to minimize non-specific adsorption of the binding agent.A "passivated" surface refers to a surface that has been treated with an outer layer of material to minimize non-specific binding of the binding agent. Methods for passivating the surface include standard methods from the fluorescence single molecule analysis literature, including passivating the surface with polymers such as polyethylene glycol (PEG) (Pan et al., 2015, Phys. Biol. 12:045006), polysiloxanes (e.g., Pluronic F-127), star polymers (e.g., star PEG) (Groll et al., 2010, Methods Enzymol. 472:1-18), hydrophobic dichlorodimethylsilane (DDS) + self-assembled Tween-20 (Hua et al., 2014, Nat. Methods 11:1233-1236) and diamond-like carbon (DLC), DLC + PEG (Stavis et al., 2011, Proc. Natl. Acad. Sci. USA 108:983-988). In addition to covalent surface modification, a number of passivating agents can be used, including surfactants such as Tween-20, polysiloxanes in solution (Pluronic series), polyvinyl alcohol (PVA), and proteins such as BSA and casein. Alternatively, when the protein, polypeptide, or peptide is immobilized on the solid substrate, the density of the protein, polypeptide, or peptide can be titrated on the surface or in the volume of the solid substrate by incorporating competing molecules or "dummy" reactive molecules (see Fig.36A ).

[0445] In certain embodiments where multiple macromolecules are immobilized on the same solid support, the macromolecules may be appropriately spaced to reduce or prevent the occurrence of cross-binding or intermolecular events, e.g., when a binding agent binds to a first macromolecule, its encoded tag information is transferred to the recording tag associated with the adjacent macromolecule rather than the recording tag associated with the first macromolecule. To control the spacing of macromolecules (e.g., proteins, polypeptides, or peptides) on a solid support, the density of functional coupling groups (e.g., TCOs) may be titrated on the surface of the matrix (see Fig.34). In some embodiments, a plurality of macromolecules are spaced apart on the surface of the solid support or in the volume (e.g., porous support) at a distance of about 50 nm to about 500 nm, or about 50 nm to about 400 nm, or about 50 nm to about 300 nm, or about 50 nm to about 200 nm, or about 50 nm to about 100 nm. In some embodiments, a plurality of macromolecules are spaced apart on the surface of the solid support at an average distance of at least 50 nm, at least 60 nm, at least 70 nm, at least 80 nm, at least 90 nm, at least 100 nm, at least 150 nm, at least 200 nm, at least 250 nm, at least 300 nm, at least 350 nm, at least 400 nm, at least 450 nm, or at least 500 nm. In some embodiments, a plurality of macromolecules are spaced apart on the surface of the solid support at an average distance of at least 50 nm. In some embodiments, macromolecules are spaced apart on the surface or in the volume of the solid support such that, empirically, the relative frequency of intermolecular to intramolecular events is <1:10; <1:100; <1:1000; or <1:10,000. Appropriate spacing frequencies can be determined empirically using functional assays (see Example 23) and can be accomplished by dilution and / or by doping with "dummy" spacer molecules that compete for attachment sites on the substrate surface.

[0446] For example, Fig.34 As shown, PEG-5000 (MW 5000) is used to block the interstitial spaces between peptides on the surface of the matrix (e.g., the surface of the beads). In addition, the peptides are coupled to functional groups that are also linked to the PEG-5000 molecules. In a preferred embodiment, this is achieved by coupling a mixture of NHS-PEG-5000-TCO + NHS-PEG-5000-methyl to amine-derivatized beads (see Fig.34 ). The stoichiometric ratio between the two PEGs (TCO to methyl) is titrated to produce an appropriate density of functional coupling groups (TCO groups) on the surface of the substrate; the methyl-PEG is inert to coupling. The effective spacing between TCO groups can be calculated by measuring the density of TCO groups on the surface. In certain embodiments, the spacing between coupling groups (e.g., TCO) on the solid surface is at least 50 nm, at least 100 nm, at least 250 nm, or at least 500 nm. After PEG 5000-TO / methyl derivatization of the beads, the excess NH 2 The group is quenched with a reactive anhydride such as acetic acid or succinic anhydride.

[0447] VI. Record Label

[0448] At least one record label is directly or indirectly bound or co-localized to the macromolecule and attached to the solid support (see, e.g. Figure 5 ). The record label may comprise DNA, RNA, PNA, γPNA, GNA, BNA, XNA, TNA, polynucleotide analogs, or a combination thereof. The record label may be single-stranded, or partially or completely double-stranded. The record label may have a blunt end or a sticky end. In certain embodiments, after the binding agent is bound to the macromolecule, the identification information of the coding label of the binding agent is transferred to the record label to produce an extended record label. Further extension of the extended record label may be performed in a subsequent binding cycle.

[0449] The record tag can be directly or indirectly (e.g., through a linker) connected to the solid support by any means known in the art, including covalent and non-covalent interactions, or any combination thereof. For example, the record tag can be connected to the solid support by a connection reaction. Alternatively, the solid support may include a reagent or coating to facilitate direct or indirect connection of the record tag to the solid support. Strategies for immobilizing nucleic acid molecules on solid supports (e.g., beads) have been described in U.S. Patent No. 5,900,481. Steinberg et al. (2004, Biopolymers 73: 597-605); Lund et al., 1988 (Nucleic Acids Res. 16: 10861-10880); and Steinberg et al. (2004, Biopolymers 73: 597-605), each of which is incorporated herein by reference in its entirety.

[0450] In certain embodiments, the co-localization of the macromolecule (e.g., peptide) and the associated record tag is achieved by conjugating the macromolecule and the record tag to a bifunctional linker directly attached to the surface of the solid support Steinberg et al. (2004, Biopolymers 73: 597-605). In further embodiments, a trifunctional group is used to derivatize the solid support (e.g., a bead), and the resulting bifunctional group is coupled to both the macromolecule and the record tag.

[0451] Methods and reagents (eg, click chemistry reagents and photoaffinity labeling reagents) such as those described for attachment of macromolecules and solid supports can also be used to attach record tags.

[0452] In a specific embodiment, a single record label is attached to a macromolecule (e.g., a peptide), preferably by attachment of an unblocked N- or C-terminal amino acid. In another embodiment, multiple record labels are attached to the macromolecule (e.g., a protein, polypeptide, or peptide), preferably attached to a lysine residue or a peptide backbone. In some embodiments, a macromolecule (e.g., a protein or polypeptide) labeled with multiple record labels is fragmented or digested into smaller peptides, each peptide being labeled with an average of one record label.

[0453] In certain embodiments, the record tag includes an optional unique molecular identifier (UMI) that provides a unique identifier tag for each macromolecule (e.g., protein, polypeptide, peptide) associated with the UMI. The UMI can be about 3 to about 40 bases, about 3 to about 30 bases, about 3 to about 20 bases, or about 3 to about 10 bases, or about 3 to about 8 bases. In some embodiments, the UMI is about 3 bases, 4 bases, 5 bases, 6 bases, 7 bases, 8 bases, 9 bases, 10 bases, 11 bases, 12 bases, 13 bases, 14 bases, 15 bases, 16 bases, 17 bases, 18 bases, 19 bases, 20 bases, 25 bases, 30 bases, 35 bases, or 40 bases in length. The UMI can be used to deconvolute sequencing data from multiple extended record tags to identify sequence reads from individual macromolecules. In some embodiments, within the macromolecule library, each macromolecule is associated with a single record tag, each record tag comprising a unique UMI. In other embodiments, multiple copies of a record tag are associated with a single macromolecule, each copy of the record tag comprising the same UMI. In some embodiments, the UMI has a base sequence that is different from the spacer or coding sequence within the binding agent coding tag to facilitate differentiation of these components during sequence analysis.

[0454] In certain embodiments, the record tag comprises a barcode, e.g., instead of a UMI (if present). A barcode is a nucleic acid molecule of about 3 to about 30 bases, about 3 to about 25 bases, about 3 to about 20 bases, about 3 to about 10 bases, about 3 to about 10 bases, about 3 to about 8 bases in length. In some embodiments, the barcode is about 3 bases, 4 bases, 5 bases, 6 bases, 7 bases, 8 bases, 9 bases, 10 bases, 11 bases, 12 bases, 13 bases, 14 bases, 15 bases, 20 bases, 25 bases, or 30 bases in length. In one embodiment, the barcode allows for multiplexed sequencing of multiple samples or libraries. Barcodes can be used to derive partitions, fractions, compartments, samples, spatial locations, or libraries of macromolecules (e.g., peptides). Barcodes can be used to deconvolute multiplexed sequence data and identify sequence reads from a single sample or library. For example, barcoded beads can be used in methods involving sample emulsification and partitioning, such as for the purpose of partitioning the proteome.

[0455] The barcode can represent a compartment label, wherein a compartment, such as a droplet, a microwell, a physical area on a solid support, etc., is assigned a unique barcode. The association of a compartment with a specific barcode can be achieved in any manner, such as by encapsulating a single barcode bead in a compartment, such as by directly merging or adding a barcode droplet to a compartment, by directly printing or injecting a barcode reagent into a compartment, etc. The barcode reagent in the compartment is used to add a compartment-specific barcode to the macromolecule or fragment thereof in the compartment. Applied to the partitioning of proteins into compartments, the barcode can be used to map the analyzed peptide back to its original protein molecule in the compartment. This can greatly facilitate protein identification. Compartment barcodes can also be used to identify protein complexes.

[0456] In other embodiments, a plurality of compartments representing a subset of a population of compartments may be assigned a unique barcode representative of the subset.

[0457] Alternatively, the barcode can be a sample identification barcode. The sample barcode can be used for multiplex analysis of a group of samples in a single reaction vessel or fixed to a collection of a single solid matrix or solid matrix (e.g., a planar slide, a bead group contained in a single tube or container, etc.). Macromolecules from many different samples can be marked with a record tag with a sample-specific barcode, and then all samples are brought together before being fixed to a solid support, cycle binding, and record tag analysis. Alternatively, the sample can be kept separated until after the DNA encoding library is produced, and the sample barcode is attached during the PCR amplification process of the DNA encoding library, and then mixed together before sequencing. When measuring analytes (e.g., proteins) of different abundance categories, this method may be useful. For example, the sample can be separated and barcoded, and a portion is treated with a binding agent that reduces the abundance analyte, and another portion is treated with a binding agent for a higher abundance analyte. In a specific embodiment, this method helps to adjust the dynamic range of a specific protein analyte assay to the "optimal point" of the standard expression level of the protein analyte.

[0458] In certain embodiments, peptides, polypeptides or proteins from multiple different samples are labeled with record tags having sample-specific barcodes. The composite sample bar-shaped peptides, polypeptides or proteins can be mixed together before the cyclic binding reaction. In this way, a highly multiplexed alternative to digital reverse phase protein arrays (RPPA) is effectively created (Guo, Liu et al. 2012, Assadi, Lamerz et al. 2013, Akbani, Becker et al. 2014, Creighton and Huang 2015). The creation of digital RPPA-like analysis has many applications in translational research, biomarker validation, drug discovery, clinical, and precision medicine.

[0459] In certain embodiments, the record tag comprises a universal priming site, for example, a forward or 5' universal priming site. The universal priming site is a nucleic acid sequence that can be used to initiate a library amplification reaction and / or for sequencing. The universal priming site may include, but is not limited to, a priming site for PCR amplification, a flow slot adapter sequence (e.g., Illumina next generation sequencing) annealed with a complementary oligonucleotide on the flow slot surface, a sequencing priming site, or a combination thereof. The universal priming site can be about 10 bases to about 60 bases. In some embodiments, the universal priming site comprises an Illumina P5 primer (5'-AATGATACGGCGACCACCGA-3'-SEQ ID NO: 133) or an Illumina P7 primer (5'-CAAGCAGAAGACGGCATACGAGAT-3'-SEQ ID NO: 134).

[0460] In certain embodiments, the record tag comprises a spacer at its end, e.g., the 3' end. As used herein, the spacer sequence in the context of the record tag includes a spacer sequence that is identical to the spacer sequence associated with its cognate binder, or includes a spacer sequence that is complementary to the spacer sequence associated with its cognate binder. The end, e.g., the 3' spacer on the record tag allows the identification information of the cognate binder to be transferred from its encoding tag to the record tag during the first binding cycle (e.g., by primer extension or sticky end ligation through annealing of the complementary spacer sequence).

[0461] In one embodiment, the length of the spacer sequence is about 1-20 bases, about 2-12 bases, or 5-10 bases. The length of the spacer may depend on factors such as the temperature and reaction conditions of the primer extension reaction used to transfer the encoded label information to the record label.

[0462] In a preferred embodiment, the spacer sequence in the record is designed to have minimal complementarity with other regions in the record label; similarly, the spacer sequence in the coding label should have minimal complementarity with other regions in the coding label. In other words, the spacer sequence of the record label and coding label should have minimal sequence complementarity with components in the record label or coding label, such as unique molecular identifiers, barcodes (e.g., compartments, partitions, samples, spatial locations), universal primer sequences, coding sequences, cycle-specific sequences, etc.

[0463] As described for the binding agent spacer, in some embodiments, the record tags associated with the macromolecular library share a common spacer sequence. In other embodiments, the record tags associated with the macromolecular library have a binding cycle-specific spacer sequence that is complementary to the binding cycle-specific spacer sequence of its cognate binder, which is useful when using non-cascade extension record tags (see Fig.10 ).

[0464] The collection of extended record tags can be concatenated afterwards (see e.g. Fig.10). After the binding cycle is completed, the bead solid support, each containing an average of one or less than one macromolecule, each macromolecule with an extended record tag set (the extended record tag set is co-localized at the macromolecule site) is placed in an emulsion. The emulsion is formed so that each droplet is occupied by at most one bead on average. An optional assembly PCR reaction is performed in the emulsion to amplify the extended record tags co-localized with the macromolecule on the beads, and to assemble them in a colinear order by priming between different cycle-specific sequences on the separated extended record tags (Xiong, Peng et al. 2008). The emulsion is then destroyed and the assembled extended record tags are sequenced.

[0465] In another embodiment, the DNA record tag comprises a universal priming sequence (U1), one or more barcode sequences (BC), and a spacer sequence (Sp1) specific to the first binding cycle. In the first binding cycle, the binding agent uses a DNA encoding tag comprising an Sp1 complementary spacer, an encoder barcode, and an optional cycle barcode, and a second spacer element (Sp2). The practicality of using at least two different spacers is that the first binding cycle selects one of several potential DNA record tags, and a single DNA record tag is extended to produce a new Sp2 spacer element at the end of the extended DNA record tag. In the second and subsequent binding cycles, the binding agent only contains Sp2' spacers instead of Sp1'. In this way, only the single extended record tag from the first cycle is extended in subsequent cycles. In another embodiment, the second and subsequent cycles can use binding agent specific spacers.

[0466] In some implementations, the record tag includes from the 5' to 3' direction: a universal forward (or 5') priming sequence, a UMI, and a spacer sequence. In some embodiments, the record tag includes from the 5' to 3' direction: a universal forward (or 5') priming sequence, an optional UMI, a barcode (e.g., a sample barcode, a partition barcode, a compartment barcode, a spatial barcode, or any combination thereof), and a spacer sequence. In some other embodiments, the record tag includes from the 5' to 3' direction: a universal forward (or 5') priming sequence, a barcode (e.g., a sample barcode, a partition barcode, a compartment barcode, a spatial barcode, or any combination thereof), an optional UMI, and a spacer sequence.

[0467] Combinatorial approaches can be used to generate UMIs from modified DNA and PNA. In one example, UMIs can be constructed by "chemically ligating" a set of short word sequences (4-15mers) that are designed to be orthogonal to each other (Spiropulos and Heemstra 2012). A DNA template is used to guide the chemical ligation of the "word" polymers. The DNA template is composed of hybrid arms that can assemble the combinatorial template structure simply by mixing the subcomponents together in solution (see Fig.12 C). In some embodiments, there are no "spacer" sequences in the design. The size of the word space can range from 10 words to 10,000 words or more. In some embodiments, the words are selected so that they are different from each other so as not to cross-hybridize, but have relatively uniform hybridization conditions. In one embodiment, the length of the words will be on the order of 10 bases, with approximately 1000 words in the subset (this is only the total 10-mer ~ 4 10 About 0.1% of the word space = 1 million words). A collection of these words (1000 in the subset) can be concatenated together to generate a complexity of 1000 n The final combined UMI of the power. For 4 words connected together, it creates 10 12The UMI diversity of different elements. These UMI sequences will be attached to the macromolecule (peptide, protein, etc.) at the single molecule level. In one embodiment, the diversity of UMI exceeds the number of molecules of the macromolecule to which the UMI is attached. In this way, the UMI can uniquely identify the macromolecule of interest. The use of combined word UMIs facilitates reading on high error rate sequencers (e.g., nanopore sequencers, nanogap channel sequencing, etc.) because single base resolution is not required to read multi-base length words. The combined word method can also be used to generate other identity information components of record labels or encoding labels, such as compartment labels, partition barcodes, spatial barcodes, sample barcodes, encoder sequences, cycle-specific sequences, and barcodes. Methods for nanopore sequencing and DNA encoding information with error-tolerant words (codes) are known in the art (see, e.g., Kiah et al., 2015, Codes for DNA sequence profiles. IEEE International Symposium on Information Theory (ISIT); Gabrys et al., 2015, Asymmetric Lee distance codes for DNA-based storage. IEEE Symposium on Information Theory (ISIT); Laure et al., 2016, Coding in 2D: Using Intentional Dispersity to Enhance the Information Capacity of Sequence-Coded Polymer Barcodes. Angew. Chem. Int. Ed. doi: 10.1002 / anie.201605279; Yazdi et al., 2015, IEEE Transactions on Molecular, Biological and Multi-Scale Communications 1: 230-248; and Yazdi et al., 2015, Sci Rep. 5:14138, each of which is incorporated herein by reference in its entirety). Thus, in certain embodiments, the extended record tag, extended coding tag, or di-tag construct in any of the embodiments described herein comprises an identification component (e.g., a UMI, coding sequence, barcode, compartment tag, cycle-specific sequence, etc.) that is an error correction code. In some embodiments, the error correction code is selected from: Hamming code, Lie distance code, asymmetric Lie distance code, Reed-Solomon code, and Levenshtein-Tenengolts code.For nanopore sequencing, the current or ion distribution and asymmetric base calling errors are inherent to the type of nanopore and biochemistry used, and this information can be used to design more robust DNA codes using the error correction methods described above. As an alternative to using robust DNA nanopore sequencing barcodes, the current or ion distribution characteristics of the barcode sequence can be used directly (U.S. Patent No. 7,060,507, which is incorporated by reference in its entirety), avoiding DNA base calling entirely, and immediately identifying the barcode sequence by mapping back to the predicted current / distribution characteristics as described by Laszlo et al. (2014, Nat. Biotechnol. 32: 829-833, incorporated by reference in its entirety). In this article, Laszlo et al. describe the current characteristics generated by the biological nanopore, MspA, when passing different strings through the nanopore, and the ability to map and identify DNA chains by mapping the generated current characteristics back to the computer to predict possible current characteristics from a universal sequence (2014, Nat. Biotechnol. 32: 829-833). Similar concepts can be applied to DNA codes and electrical signals generated by DNA sequencing based on nanogap channel currents (Ohshiro et al., 2012, Sci Rep 2:501).

[0468] Thus, in certain embodiments, the identification component of the encoding tag, recording tag, or both is capable of generating a unique current or ion distribution or optical signature, wherein the analyzing step of any of the methods provided herein comprises detecting the unique current or ion distribution or optical signature to identify the identification component. In some embodiments, the identification component is selected from an encoder sequence, a barcode, a UMI, a compartment tag, a cycle-specific sequence, or any combination thereof.

[0469] In certain embodiments, all or a substantial amount of macromolecules (e.g., proteins, polypeptides or peptides) in a sample (e.g., at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%) are labeled with a record tag. The labeling of the macromolecules can be performed before or after the macromolecules are fixed to a solid support.

[0470] In other embodiments, a subset of macromolecules (e.g., proteins, polypeptides, or peptides) within a sample are labeled with a record tag. In a specific embodiment, a subset of macromolecules from a sample are targeted (analyte-specific) labeled with a record tag. Targeted record tagging of proteins can be achieved using a target protein-specific binding agent (e.g., an antibody, an aptamer, etc.) that is linked to a short target-specific DNA capture probe, e.g., an analyte-specific barcode, that is annealed to a complementary target-specific bait sequence in the record tag, e.g., an analyte-specific barcode (see Fig.28 A). The recording tag comprises a reactive group for a homologous reactive group present on the target protein (e.g., click chemistry tag, photoaffinity tag). For example, the recording tag may comprise an azide group for interaction with an alkyne-derivatized protein, or the recording tag may comprise a benzophenone for interaction with a native protein, etc. (see Fig.28 AB). After the target protein is bound by the target protein-specific binding agent, the recording tag and the target protein are coupled via their corresponding reactive groups (see Fig.28 BC). After labeling the target protein with a recording tag, the target protein-specific binding agent can be removed by digesting the DNA capture probe attached to the target protein-specific binding agent. For example, the DNA capture probe can be designed to contain uracil bases, which are then removed by uridine-specific removal reagents (e.g., USER TM ) is subjected to targeted digestion, and the target protein-specific binding agent can be dissociated from the target protein.

[0471] In one example, a DNA capture probe (e.g., Fig.28 Analyte barcodes in BC A ) is labeled with an antibody specific for a target protein set, and the DNA capture probe is designed with a complementary bait sequence (e.g., Fig.28 Analyte barcodes in BC A' Sample-specific labeling of proteins can be achieved by using antibodies labeled with DNA capture probes to hybridize with complementary bait sequences on the record tags containing sample-specific barcodes.

[0472] In another example, a target protein-specific aptamer is used for targeted record labeling of a subset of proteins in a sample. The target-specific aptamer is connected to a DNA capture probe that anneals to a complementary bait sequence in the record label. The record label contains a reactive chemical probe or a photoreactive chemical probe (e.g., benzophenone (BP)) for coupling to the target protein with a corresponding reactive group. The aptamer binds to its target protein molecule, bringing the record label close to the target protein so that the record label is coupled to the target protein.

[0473] Labeling of proteins using photoaffinity (PA) photoreactive chemical probes attached to small molecule protein affinity ligands has been previously described (Park, Koh et al. 2016). Typical photoreactive chemical probes include probes based on benzophenone (reactive diradical, 365 nm), phenyldiazirine (reactive carbon, 365 nm), and phenylazide (reactive nitrene radical, 260 nm), activated under irradiation wavelengths as described previously (Smith and Collins 2015). In a preferred embodiment, the target protein in a protein sample is labeled with a record tag containing a sample barcode using the method disclosed by Li et al., wherein the bait sequence in the benzophenone-labeled record tag is attached to a cognate binder (e.g., a nucleic acid aptamer (see Fig.28 ) DNA capture probe hybridization (Li, Liu et al, 2013). For photoaffinity labeled protein targets, the use of DNA / RNA aptamers as target protein specific binders is superior to antibodies because the photoaffinity group can self-label the antibody instead of the target protein. In contrast, the efficiency of photoaffinity labeling of nucleic acids is lower than that of labeling proteins, which makes aptamers a better carrier for DNA-guided chemical or optical labeling. Similar to photoaffinity labeling, DNA-guided chemical labeling of reactive lysines (or other groups) near the aptamer binding site can also be used in a manner similar to that described by Rosen et al. (Rosen, Kodal et al. 2014, Kodal, Rosen et al. 2016).

[0474] In the foregoing embodiments, other types of linkage besides hybridization may be used to link the target-specific binding agent and the recording label (see Fig.28 A) For example, Fig.28 As shown in Figure B, once the captured target protein (or other macromolecule) is covalently attached to the record tag, a linker designed to cleave and release the binding agent can be used to covalently link the two groups. Suitable linkers can be attached to multiple positions of the record tag, such as the 3' end, or to a linker attached to the 5' end of the record tag.

[0475] VII. Binders and Coding Tags

[0476] The methods described herein use a binding agent that can bind to the macromolecule. A binding agent can be any molecule (e.g., peptides, polypeptides, proteins, nucleic acids, carbohydrates, small molecules, and the like) that can bind to a component or feature of a macromolecule. 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 connected subunits of a macromolecule (e.g., a dipeptide, a tripeptide, or a higher order peptide of a longer peptide molecule).

[0477] In certain embodiments, the binding agent can be designed to covalently bind. Covalent binding can be designed to be conditional or preferred when combined with the correct group. For example, NTAA and its homologous NTAA specific binding agent can be modified with reactive groups respectively, so that once the NTAA specific binding agent is combined with the homologous NTAA, a coupling reaction is performed to produce a covalent connection between the two. The non-specific binding of the binding agent to other positions lacking a homologous reactive group will not result in covalent bonding. The covalent bonding between the binding agent and its target allows more stringent washing to be used to remove the non-specifically bound binding agent, thereby increasing the specificity of the assay.

[0478] In certain embodiments, the binding agent can be a selective binding agent. As used herein, selective binding refers to the ability of the binding agent to preferentially bind to a specific ligand (e.g., amino acid or amino acid class) relative to binding to different ligands (e.g., amino acids or amino acids). Selectivity generally refers to the equilibrium constant of a reaction in which a ligand is replaced by another ligand in a complex with a binding agent. Typically, this selectivity is related to the spatial geometry of the ligand and / or the manner and degree to which the ligand is bound to the binding agent, such as by hydrogen bonds or van der Waals forces (non-covalent interactions) or by reversible or irreversible covalent attachment to the binding agent. It should also be understood that selectivity can be relative and opposite to absolute, and it can be affected by different factors including ligand concentration. Therefore, in one example, the binding agent selectively binds to one of the twenty standard amino acids. In an example of non-selective binding, the binding agent can bind to two or more of the twenty standard amino acids.

[0479] In the practice of the methods disclosed herein, the ability of a binding agent to selectively bind to a feature or component of a macromolecule need only be sufficient to allow transfer of its coded tag information to the recorded tag associated with the macromolecule, transfer of the recorded tag information to the coded tag, or transfer of the coded tag information and the recorded tag information to the di-tag molecule. Therefore, selectivity need only be relative to other binding agents exposed to the macromolecule. It should also be understood that the selectivity of the binding agent need not be absolutely for a specific amino acid, but can be selective for a class of amino acids, such as amino acids with non-polar or non-polar side chains, or amino acids with charged (positive or negative) side chains, or amino acids with aromatic side chains, or amino acids with side chains of certain specific classes or sizes, and the like.

[0480] In a specific embodiment, the binding agent has high affinity and high selectivity for the target macromolecule. In particular, a high binding affinity with a low dissociation rate is effective for information transfer between the coding tag and the recording tag. In certain embodiments, the binding agent has a Kd of <10nM, <5nM, <1nM, <0.5nM or <0.1nM. In a specific embodiment, the binding agent is added to the macromolecule at a concentration of >10X, >100X or >1000X to allow it to bind to completion. Chang et al. describe a detailed discussion of the binding kinetics of antibodies to single protein molecules (Chang, Rissin et al. 2012).

[0481] To increase the affinity of the binder for the small N-terminal amino acid (NTAA) of the peptide, the NTAA can be modified with an "immunogenic" hapten, such as dinitrophenol (DNP). The Sanger reagent, dinitrofluorobenzene (DNFB), can be used to implement the cycle sequencing method, which attaches the DNP group to the amine group of the NTAA. Commercially available anti-DNP antibodies have affinities in the low nM range (~8nM, LO-DNP-2) (Bilgicer, Thomas et al. 2009); therefore, it is reasonable to believe that high-affinity NTAA binders can be made into many NTAAs modified with DNP (via DNFB) while achieving good binding selectivity for specific NTAAs. In another example, NTAA can be modified with sulfonyl nitrophenol (SNP) by 4-sulfonyl-2-nitrofluorobenzene (SNFB). Similar affinity enhancements can also be achieved with alternative NTAA modifiers such as acetyl or amidino (guanidino).

[0482] In certain embodiments, the binding agent can bind to NTAA, CTAA, intervening amino acids, dipeptides (sequences of two amino acids), tripeptides (sequences of three amino acids), or higher order peptides of a peptide molecule. In some embodiments, each binding agent in the binding agent library selectively binds to a specific amino acid, such as one of the twenty standard naturally occurring amino acids. Standard natural 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).

[0483] In certain embodiments, the binding agent may bind to a post-translational modification of an amino acid. In some embodiments, the peptide comprises one or more identical or different post-translational modifications. The NTAA, CTAA, intervening amino acid, or a combination thereof may be post-translationally modified. Post-translational modifications of amino acids include acylation, acetylation, alkylation (including methylation), biotinylation, butyrylation, carbamylation, carbonylation, deamidation, deamidation, dinaphthylamine formation, disulfide bond formation, elimination, flavin attachment, formylation, γ-carboxylation, glutamylation, glycylation, glycosylation, glypiation, heme C attachment, hydroxylation, hypusine formation, iodination, isoprenylation, lipidation, lipoylation, malonylation, methylation, myristoylation, oxidation, palmitoylation, pegylation, phosphate, phosphorylation, isoprenylation, propionylation, retinylidene Schiff base formation, S-glutathionylation, S-nitrosylation, S-sulfenylation, selenylation, succinylation, sulfation, ubiquitination, and C-terminal amidation (see also, Seo and Lee, 2004, J. Biochem. Mol. Biol. 37:35-44).

[0484] In certain embodiments, lectins are used as binding agents for detecting the glycosylation state of proteins, polypeptides or peptides. Lectins are carbohydrate binding proteins that can selectively recognize free carbohydrates or glycan epitopes of glycoproteins. The list of lectins that recognize various glycosylation states (e.g., core-fucose, sialic acid, N-acetyl-D-lactosamine, mannose, N-acetyl-glucosamine) includes: A, AAA, AAL, ABA, ACA, ACG, ACL, AOL, ASA, BanLec, BC2L-A, BC2LCN, BPA, BPL, Calsepa, CGL2, CNL, Con, ConA, DBA, Discoidin, DSA, ECA, EEL, F17AG, Gal1, Gal1-S, Gal2, Gal3, Gal3C-S, Gal7-S, Gal9, GNA, GRFT, GS-1, GS-II, GSL-1, GSL-II, HHL, HIHA, HPA, I, II, Jacal in, LBA, LCA, LEA, LEL, Lentil, Lotus, LSL-N, LTL, MAA, MAH, MAL_I, Malectin, MOA, MPA, MPL, NPA, Orysata, PA-IIL, PA-IL, PALa, PHA-E, PHA-L, PHA-P, PHAE, PHAL, PNA, PPL, PSA, PSL1a, PTL, PTL-1, PWM, RCA120, RS-Fuc, SAMB, SBA, SJA, SNA, SNA-1, SNA-II, SSA, STL, TJA-I, TJA-II, TxLCI, UDA, UEA-1, UEA-II, VFA, VVA, WFA, WGA (see, Zhanget al., 2016, MABS 8:524-535).

[0485] In certain embodiments, the binding agent can be combined with a modified or labeled NTAA. The modified or labeled NTAA can be a NTAA labeled with one of PITC, 1-fluoro-2,4-dinitrobenzene (Sanger reagent, DNFB), dansyl chloride (DNS-Cl, or 1-dimethylaminonaphthalene-5-sulfonyl chloride), 4-sulfonyl-2-nitrofluorobenzene (SNFB), an acetylation agent, a guanidation agent, a thioacylation agent, a thioacetylation agent, or a thiobenzylation agent.

[0486] In certain embodiments, the binding agent can be an aptamer (e.g., a peptide aptamer, a DNA aptamer, or an RNA aptamer), an antibody, anticalin, ATP-dependent Clp protease adaptor protein (ClpS), an antibody binding fragment, an antibody mimetic, a peptide, a peptide mimetic, a protein, or a polynucleotide (e.g., DNA, RNA, peptide nucleic acid (PNA), γPNA, bridging nucleic acid (BNA), xenogeneic nucleic acid (XNA), glycerol nucleic acid (GNA), or threose nucleic acid (TNA), or variants thereof).

[0487] As used herein, the terms antibody and antibody are used in a broad sense and include not only complete antibody molecules, such as but not limited to immunoglobulin A, immunoglobulin G, immunoglobulin, immunoglobulin E, and immunoglobulin M, but also immunologically active components of antibody molecules that specifically bind to at least one epitope. Antibodies can be naturally occurring, synthetically produced, or recombinantly expressed. Antibodies can be fusion proteins. Antibodies can be antibody mimetics. Examples of antibodies include, but are not limited to, Fab fragments, Fab' fragments, F(ab') 2 Fragments, single-chain antibody fragments (scFv), small antibodies, diabodies, cross-linked antibody fragments, Affibody TM , nanobodies, single domain antibodies, DVD-Ig molecules, alpha bodies, affimers, affitins, cyclotides, molecules, and the like. Immunoreaction products derived using antibody engineering or protein engineering techniques are also clearly within the meaning of the term antibody. Detailed descriptions of antibody and / or protein engineering, including related protocols, can be found in J. Maynard and G. Georgiou, 2000, Ann. Rev. Biomed. Eng. 2: 339-76; Antibody Engineering, R. Kontermann and S. Dubel, eds., Springer Lab Manual, Springer Verlag (2001); US Patent No. 5,831,012; and S. Paul, Antibody Engineering Protocols, Humana Press (1995), among other places.

[0488] As with antibodies, nucleic acid and peptide aptamers that specifically recognize peptides can be produced using known methods. Although aptamers generally bind target molecules with high affinity, highly specificity, and conformation-dependently, aptamers with lower binding affinity can be selected. Aptamers have been shown to distinguish targets based on very small structural differences, such as the presence or absence of methyl or hydroxyl groups, and some aptamers can distinguish between D- and L-enantiomers. Aptamers that bind to small molecule targets, including drugs, metal ions and organic dyes, peptides, biotin, and proteins, including but not limited to streptavidin, VEGF, and viral proteins, have been obtained. Aptamers have been shown to retain functional activity after biotinylation, fluorescein labeling, and when attached to glass surfaces and microspheres (see, Jayasena, 1999, Clin Chem 45: 1628-50; Kusser 2000, J. Biotechnol. 74: 27-39; Colas, 2000, Curr Opin Chem Biol 4: 54-9). Aptamers that specifically bind to arginine and AMP have been described (see, Patel and Suri, 2000, J. Biotech. 74:39-60). Oligonucleotide aptamers that bind to specific amino acids have been disclosed by Gold et al. (1995, Ann. Rev. Biochem. 64:763-97). RNA aptamers that bind to amino acids have also been described (Ames and Breaker, 2011, RNA Biol. 8; 82-89; Mannironi et al., 2000, RNA 6:520-27; Famulok, 1994, J. Am. Chem. Soc. 116:1698-1706).

[0489] Binding agents can be prepared by genetically modifying naturally occurring or synthetically produced proteins to introduce one or more mutations in the amino acid sequence to produce engineered proteins (e.g., NTAA, CTAA, or post-translationally modified amino acids or peptides) that bind to specific components or features of macromolecules. For example, exopeptidases (e.g., aminopeptidases, carboxypeptidases), exoproteinases, mutated exoproteinases, mutated anticalins, mutated ClpS, antibodies, or tRNA synthetases can be modified to produce binding agents that selectively bind to specific NTAAs. In another example, carboxypeptidases can be modified to produce binding agents that selectively bind to specific CTAAs. The binder can also be designed, modified, and applied to specifically bind to a modified NTAA or modified CTAA, such as a CTAA with a post-translational modification (e.g., phosphorylated NTAA or phosphorylated CTAA), or a CTAA modified by a tag (e.g., PTC, 1-fluoro-2,4-dinitrobenzene (using Sanger reagent, DNFB), dansyl chloride ((using DNS-Cl, or 1-dimethylaminonaphthalene-5-sulfonyl chloride), or using a thioacylation agent, a thioacetylation agent, an acetylation agent, an amidation (guanidation) agent, or a thiobenzylation agent). Strategies for directed protein evolution are known in the art (e.g., reviewed by Yuan et al., 2005, Microbiol. Mol. Biol. Rev. 69: 373-392), including phage display, ribosome display, mRNA display, CIS display, CAD display, emulsification, cell surface display, yeast surface display, bacterial surface display, and the like.

[0490] In some embodiments, a binding agent that selectively binds to the modified NTAA can be used. For example, the NTAA can be reacted with phenyl isothiocyanate (PITC) to form a phenylthiocarbamoyl-NTAA derivative. In this way, the binding agent can be made to selectively bind both the benzene group of the phenylthiocarbamoyl group and the α-carbon R group of the NTAA. As described below, the use of PITC in this manner allows for the subsequent cleavage of the NTAA by Edman degradation. In another embodiment, the NTAA can be reacted with Sanger reagent (DNFB) to produce DNP-labeled NTAA (see Figure 3). Optionally, DNFB is used with an ionic liquid, such as 1-[ethyl-3-methylimidazolium bis[(trifluoromethyl)sulfonyl]imide ([emim][Tf2N]), in which DNFB is highly soluble. In this way, the binding agent can be modified to selectively bind to the combination of the DNP and R groups on the NTAA. The addition of the DNP group provides a larger "handle" for the interaction of the binding agent with the NTAA and should produce a higher affinity interaction. In another embodiment, the binding agent can be an aminopeptidase that has been engineered to recognize the DNP-labeled NTAA, thereby providing a cyclic control for aminopeptidase degradation of the peptide. Once the DNP-labeled NTAA is cleaved, another round of DNFB derivatization is performed to bind and cleave the newly exposed NTAA. In a preferred embodiment, the aminopeptidase is a monomeric metalloprotease, such as an aminopeptidase activated by zinc (Calcagno and Klein 2016). In another example, the binding agent can selectively bind to NTAA modified with sulfonyl nitrophenol (SNP), such as by using 4-sulfonyl-2-nitrofluorobenzene (SNFB). In another embodiment, the binding agent can selectively bind to acetylated or amidated NTAA.

[0491] Other reagents that can be used to modify the NTAA include trifluoroethyl isothiocyanate, allyl isothiocyanate, and dimethylaminoazobenzene isothiocyanate.

[0492] Binders can be engineered for high affinity modified NTAAs, high specificity modified NTAAs, or both. In some embodiments, binders can be developed by directed evolution of well-performing affinity scaffolds using phage display.

[0493] Engineered aminopeptidase mutants that bind and cleave single or small groups of labeled (biotinylated) NTAAs have been described (see PCT Publication No. WO2010 / 065322, which is incorporated by reference in its entirety). Aminopeptidases are enzymes that cleave amino acids from the N-terminus of a protein or peptide. Natural aminopeptidases have very limited specificity and cleave the N-terminal amino acids one by one in a progressive manner (Kishor et al., 2015, Anal. Biochem. 488: 6-8). However, residue-specific aminopeptidases have been identified (Eriquez et al., J. Clin. Microbiol. 1980, 12: 667-71; Wilce et al., 1998, Proc. Natl. Acad. Sci. USA 95: 3472-3477; Liao et al., 2004, Prot. Sci. 13: 1802-10). Aminopeptidases can be engineered to specifically bind to 20 different NTAAs, which represent standard amino acids labeled with specific groups (e.g., PTC, DNP, SNP, etc.). The progressive degradation of the N-terminus of a peptide is controlled by using an engineered aminopeptidase that is active (e.g., binding activity or catalytic activity) only when the label is present. In another example, Havranak et al. (U.S. Patent Publication 2014 / 0273004) describe engineered aminoacyl tRNA synthetases (aaRS) as specific NTAA binders. The amino acid binding pocket of the aaRS can naturally bind to homologous amino acids, but generally exhibits weak binding affinity and specificity. Moreover, these natural amino acid binders do not recognize N-terminal tags. The directed evolution of aaRS scaffolds can be used to produce higher affinity, higher specificity binders that recognize the N-terminal amino acid in the context of an N-terminal tag.

[0494] In another example, highly selective engineered ClpS has also been described in the literature. Emili et al. described the directed evolution of the E. coli ClpS protein by bacterial display, generating four different variants with the ability to selectively bind NTAA to aspartate, arginine, tryptophan, and leucine residues (U.S. Pat. No. 9,566,335, the entire contents of which are incorporated by reference).

[0495] In a specific embodiment, anticalins are engineered to have high affinity and high specificity for labeled NTAAs (e.g., DNP, SNP, acetylation, etc.). Certain varieties of anticalin scaffolds have a shape suitable for binding to a single amino acid by virtue of their β-barrel structure. The N-terminal amino acid (with or without modification) may fit and be recognized in this "β-barrel" barrel. High-affinity anticalins with engineered new binding activity have been described (reviewed by Skerra, 2008, FEBS J. 275: 2677-2683). For example, anticalins with high affinity have been designed to bind (low nM) to fluorescein and digoxigenin (Gebauer and Skerra 2012). Banta et al. also reviewed the engineering of alternative scaffolds for new binding functions. (2013, Annu. Rev. Biomed. Eng. 15: 93-113).

[0496] The functional affinity (avidity) of a given monovalent binder can be increased by at least an order of magnitude by using divalent or higher order multimers of a monovalent binder (Vauquelin and Charlton 2013). Affinity refers to the cumulative strength of multiple simultaneous non-covalent binding interactions. Individual binding interactions can dissociate easily. However, when multiple binding interactions are present simultaneously, the instantaneous dissociation of a single binding interaction does not allow the binding protein to diffuse and the binding interaction to recover. Another way to increase the affinity of a binder is to include complementary sequences in the encoding tag attached to the binder and the recording tag associated with the macromolecule.

[0497] In some embodiments, a binding agent that selectively binds to a modified C-terminal amino acid (CTAA) can be used. Carboxypeptidases are proteases that cut terminal amino acids containing free carboxyl groups. Many carboxypeptidases show amino acid preferences, for example, carboxypeptidase B preferentially cuts on basic amino acids such as arginine and lysine. Carboxypeptidases can be modified to produce binding agents that selectively bind to specific amino acids. In some embodiments, carboxypeptidases can be transformed to selectively bind to modified portions and -carbon R groups of CTAA. Therefore, engineered carboxypeptidases can specifically recognize 20 different CTAAs representing standard amino acids in the background of C-terminal tags. By using engineered carboxypeptidases that are active only in the presence of a tag (e.g., binding activity or catalytic activity) to control the progressive degradation from the C-terminus of a peptide. In one example, CTAA can be modified by p-nitroaniline or 7-amino-4-methylcoumaryl.

[0498] Other potential scaffolds that can be designed to generate adhesives for the methods described herein include: anticalins, amino acid tRNA synthetases (aaRS), ClpS, Adnectin TM , T cell receptor, zinc finger protein, thioredoxin, GST A1-1, DARPin, affimer, affitin, alphabody, avimer, Kunitz domain peptide, monomer, single domain antibody, EETI-II, HPSTI, intracellular antibody, lipocalin, PHD finger, V(NAR)LDTI, evibody, Ig(NAR), knottin, maxibody, neocarzinostatin, pVIII, tendamistat, VLR, protein A scaffold, MTI-II, ecotin, GC N4, Im9, kunitz domain, microbody, PBP, transbody, tetranectin, WW domain, CBM4-2, DX-88, GFP, iMab, Ldl receptor domain A, Min-23, PDZ domain, avian pancreatic polypeptide, charybdotoxin / 10Fn3, domain antibody (Dab), a2p8 ankyrin repeat, insect defense A peptide, designed AR protein, C-type lectin domain, staphylococcal nuclease, Src homology domain 3 (SH3), or Src homology domain 2 (SH2).

[0499] Binders can be designed to withstand higher temperatures and mild denaturation conditions (e.g., in the presence of urea, guanidine thiocyanate, ionic solutions, etc.). The use of denaturants helps reduce secondary structures in surface-bound peptides, such as -helices, hairpins, -strands, and other such structures, which may interfere with binding of the binder to linear peptide epitopes. In one embodiment, ionic liquids such as 1-ethyl-3-methylimidazolium acetate ([EMIM]+[ACE]) are used to reduce peptide secondary structure during the binding cycle (Lesch, Heuer et al. 2015).

[0500] Any described binding agent also comprises a coding tag containing identification information about the binding agent. The coding tag is a nucleic acid molecule of about 3 bases to about 100 bases, which provides unique identification information for its related binding agent. The coding tag can comprise about 3 to about 90 bases, about 3 to about 80 bases, about 3 to about 70 bases, about 3 to about 60 bases, about 3 bases to about 50 bases, about 3 bases to about 40 bases, about 3 bases to about 30 bases, about 3 bases to about 20 bases, about 3 bases to about 10 bases, or about 3 bases to about 8 bases. In some embodiments, the coding tag is about 3 bases, 4 bases, 5 bases, 6 bases, 7 bases, 8 bases, 9 bases, 10 bases, 11 bases, 12 bases, 13 bases, 14 bases, 15 bases, 16 bases, 17 bases, 18 bases, 19 bases, 20 bases, 25 bases, 30 bases, 35 bases, 40 bases, 55 bases, 60 bases, 65 bases, 70 bases, 75 bases, 80 bases, 85 bases, 90 bases, 95 bases, or 100 bases. The coding tag can be composed of DNA, RNA, polynucleotide analogs, or a combination thereof. Polynucleotide analogs include PNA, gamma PNA, BNA, GNA, TNA, LNA, morpholino polynucleotides, 2'-O-methyl polynucleotides, alkylribosyl substituted polynucleotides, phosphorothioate polynucleotides, and 7-deazapurine analogs.

[0501] The coding tag comprises an encoder sequence, which provides identification information about the related binding agent. The encoding sequence is about 3 bases to about 30 bases, about 3 bases to about 20 bases, about 3 bases to about 10 bases, or about 3 bases to about 8 bases. In some embodiments, the encoding sequence is about 3 bases, 4 bases, 5 bases, 6 bases, 7 bases, 8 bases, 9 bases, 10 bases, 11 bases, 12 bases, 13 bases, 14 bases, 15 bases, 20 bases, 25 bases or 30 bases in length. The length of the encoder sequence determines the quantity of the unique encoder sequence that can be generated. Shorter encoding sequences produce a smaller number of unique encoding sequences, which may be useful when using a small amount of binding agent. When analyzing a group of macromolecules, longer encoder sequences may be required. For example, a 5 base coding sequence has the formula 5'-NNNNN-3' (SEQ ID NO: 135), where N can be any naturally occurring nucleotide or analog. Using the four naturally occurring nucleotides A, T, C, and G, the total number of unique coding sequences having a length of 5 bases is 1,024. In some embodiments, the total number of unique coding sequences can be reduced by excluding, for example, encoder sequences where all bases are identical, at least three consecutive bases are identical, or both. In a particular embodiment, a set of ≥50 unique encoder sequences is used for a library of binders.

[0502] In some embodiments, identifying a component of a coding tag or record tag, such as an encoder sequence, a barcode, a UMI, a compartment label, a partition barcode, a sample barcode, a spatial region barcode, a cycle-specific sequence, or any combination thereof, is subject to a Hamming distance, a Lee distance, an asymmetric Lee distance, Reed-Solomon, a Levenshtein-Tenengolts, or a similar error correction method. The Hamming distance refers to the number of positions that are different between two strings of equal length. It measures the minimum number of substitutions required to change one string to another. The Hamming distance can be used to correct errors by selecting encoder sequences that are at a reasonable distance. Thus, in the example where the encoder sequence is 5 bases, the number of encoder sequences is reduced to 256 unique encoder sequences (Hamming distance 1→4 4encoder sequences = 256 encoder sequences). In another embodiment, the encoder sequence, barcode, UMI, compartment tag, cycle-specific sequence, or any combination thereof is designed to be easily read out by a cycle decoding process (Gunderson, 2004, Genome Res. 14: 870-7). In another embodiment, the encoder sequence, barcode, UMI, compartment tag, partition barcode, spatial barcode, sample barcode, cycle-specific sequence, or any combination thereof is designed to be read out by low-precision nanopore sequencing because single base resolution is not required, but rather words of composite bases (about 5-20 bases in length) need to be read. A subset of 15-mer, error-corrected Hamming barcodes that can be used in the methods of the present disclosure are shown in SEQ ID NOs: 1-65, and their corresponding reverse complement sequences are shown in SEQ ID NOs: 66-130.

[0503] In some embodiments, every kind of unique binding agent in the binding agent library has a unique coding sequence.For example, 20 kinds of unique coding sequences can be used for the library of 20 kinds of binding agents in conjunction with 20 kinds of standard amino acids.Extra coding tag sequences can be used for identifying modified amino acids (for example, post-translationally modified amino acids).In another example, 30 kinds of unique coding sequences can be used for the library of 30 kinds of binding agents in conjunction with the 20 kinds of standard amino acids and 10 kinds of post-translationally modified amino acids (for example phosphorylated amino acids, acetylated amino acids, methylated amino acids).In other embodiments, two or more different binding agents can share the same encoder sequence.For example, two kinds of binding agents in conjunction with different standard amino acids can share the same coding sequence.

[0504] In certain embodiments, the coding tag also includes a spacer sequence at one or both ends. The spacer sequence is about 1 base to about 20 bases, about 1 base to about 10 bases, about 5 bases to about 9 bases, or about 4 bases to about 8 bases. In some embodiments, the length of the spacer is about 1 base, 2 bases, 3 bases, 4 bases, 5 bases, 6 bases, 7 bases, 8 bases, 9 bases, 10 bases, 11 bases, 12 bases, 13 bases, 14 bases, 15 bases, or 20 bases. In some embodiments, the spacer within the coding tag is shorter than the coding sequence, for example, at least 1 base, 2 bases, 3 bases, 4 bases, 5 bases, 6 bases, 7 bases, 8 bases, 9 bases, 10 bases, 11 bases, 12 bases, 13 bases, 14 bases, 15 bases, 20 bases or 25 bases. In other embodiments, the spacer within the coding tag is the same length as the encoder sequence. In certain embodiments, the spacer is binding agent specific, so that the spacer from the previous binding cycle only interacts with the spacer from the suitable binding agent in the current binding cycle. An example is a homologous antibody pair containing a spacer sequence, which allows information transfer only when the two antibodies bind to the macromolecule sequentially. The spacer sequence can be used as a primer annealing site for a primer extension reaction, or a splint or sticky end in a ligation reaction. The 5' spacer region on the coding tag (see Figure 5 A, "*Sp'") may optionally contain pseudo-complementary bases to the 3' spacer on the recording label to increase Tm (Lehoud et al., 2008, Nucleic Acids Res. 36:3409-3419).

[0505] In some embodiments, the encoding tags within a collection of binders share a common spacer sequence used in an assay (e.g., an entire library of binders used in a multiplexed binding cycle method has a common spacer in their encoding tags). In another embodiment, the encoding tags comprise a binding cycle tag that identifies a specific binding cycle. In other embodiments, the encoding tags within the library of binders have a binding cycle-specific spacer sequence. In some embodiments, the encoding tags comprise one binding cycle-specific spacer sequence. For example, the encoding tags for binders used in the first binding cycle comprise a "cycle 1"-specific spacer sequence, the encoding tags for binders used in the second binding cycle comprise a "cycle 2"-specific spacer sequence, and so on up to "n" binding cycles. In a further embodiment, the encoding tags for binders used in the first binding cycle comprise a "cycle 1"-specific spacer sequence and a "cycle 2"-specific spacer sequence, the encoding tags for binders used in the second binding cycle include a "cycle 2"-specific spacer sequence and a "cycle 3"-specific spacer sequence, and so on up to "n" binding cycles. This embodiment can be used for subsequent PCR assembly of non-cascade extension record tags after the binding cycles are completed (see Fig.10 ). In some embodiments, the spacer sequence comprises a sufficient number of bases to anneal to a complementary spacer sequence in a recording tag or an extended recording tag to initiate a primer extension reaction or a sticky end ligation reaction.

[0506] When a group of record tags are associated with a macromolecule, cycle-specific spacer sequences can also be used to cascade the information of the coded tags to a single record tag. The first binding cycle transfers the information from the coded tag to a randomly selected record tag, and subsequent binding cycles can trigger only the extended record tag through a cycle-dependent spacer sequence. More specifically, the coded tag of the binder used in the first binding cycle includes a "cycle 1" specific spacer sequence and a "cycle 2" specific spacer sequence, and the coded tag of the binder used in the second binding cycle includes a "cycle 2" specific spacer sequence and a "cycle 3" specific spacer sequence, and so on to "n" binding cycles. The coded tag of the binder from the first binding cycle can anneal with the record tag through a complementary cycle 1 specific spacer sequence. When the coded tag information is transferred to the record tag, the cycle 2 specific spacer is located at the 3' end of the extended record tag at the end of the 1. The coded tag of the binder from the second binding cycle can anneal with the extended record tag through a complementary cycle 2 specific spacer sequence. When the coding tag information is transferred to the extended record tag, the cycle 3 specific spacer sequence is located at the 3' end of the extended record tag at the end of the binding cycle 2, and so on to complete the "n" binding cycle. This embodiment stipulates that the transfer of binding information in a specific binding cycle between multiple binding cycles will only occur on the (extended) record tag that has undergone the previous binding cycle. However, sometimes the binding agent will not be able to bind to the homologous macromolecule. Oligonucleotides containing binding cycle specific spacers after each binding cycle can be used as a "tracking" step to keep the binding cycle synchronized, even if the binding cycle event fails. For example, if the homologous binder cannot bind to the macromolecule during binding cycle 1, then after binding cycle 1, an oligonucleotide containing cycle 1 specific spacers, cycle 2 specific spacers and "invalid" coding sequences is used to increase the tracking step. The "invalid" encoder sequence can lack an encoder sequence, or preferably, it is a specific barcode that positively identifies the "invalid" binding cycle. The "invalid" oligonucleotide can anneal with the record tag through the cycle 1 specific spacer, and the cycle 2 specific spacer is transferred to the record tag. Thus, despite the failed binding cycle 1 event, binders from binding cycle 2 are still able to anneal to the extended record tag via the cycle 2 specific spacer. The "null" oligonucleotide marks binding cycle 1 as a failed binding event within the extended record tag.

[0507] In a preferred embodiment, a binding cycle-specific encoder sequence is used to encode the tag. The binding cycle-specific encoder sequence can be used by using a completely unique analyte binding cycle encoder barcode (e.g., NTAA) or in combination with a link to a cycle-specific barcode (see Fig.35) analyte (e.g., NTAA) encoder sequence. The advantage of using a combinatorial approach is that fewer total barcodes need to be designed. For a group of 20 analyte binders for 10 cycles, only 20 analyte encoder sequence barcodes and 10 binding cycle specific barcodes need to be designed. On the contrary, if the binding cycle is directly embedded in the binding agent encoder sequence, a total of 200 independent encoder barcodes may need to be designed. The advantage of directly embedding the binding cycle information in the encoder sequence is that when error correction barcodes are used on nanopore readout, the total length of the encoded label can be minimized. The use of error-tolerant barcodes allows the use of sequencing platforms and methods that are more prone to errors to perform highly accurate barcode identification, but has other advantages, such as rapid analysis, lower cost, and / or more portable instruments. One such example is based on nanopore sequencing readout.

[0508] In some embodiments, the coding tag comprises a cleavable or nickable DNA strand within the second (3') spacer sequence near the binding agent (see Figure 32). For example, the 3' spacer may have one or more uracil bases that can be nicked by a uracil-specific excision reagent (USER). USER produces a single nucleotide gap at the position of the uracil. In another example, the 3' spacer may comprise a recognition sequence for a nicking endonuclease that hydrolyzes only one strand of the duplex. Preferably, the enzyme used to cut or nick the 3' spacer sequence acts only on one DNA strand (the 3' spacer of the coding tag), so that the other strand within the duplex belonging to the (extended) recording tag remains intact. These embodiments are particularly useful in the analysis of proteins in their native conformation because it allows the binding agent to be removed from the (extended) recording tag without denaturation after primer extension occurs, and leaves a single-stranded DNA spacer sequence on the extended recording tag that can be used for subsequent binding cycles.

[0509] The coding tag can also be designed to include a palindromic sequence. Including a palindromic sequence in the coding tag allows the newly grown, extended recording tag to fold itself when the coding tag information is transferred. The extended recording tag folds into a more compact structure, effectively reducing undesirable intermolecular binding and primer extension events.

[0510] In some embodiments, the coding tag comprises an analyte-specific spacer that can cause extension only on a record tag extended with a binding agent that previously identified the same analyte. The coding tag comprising an analyte-specific spacer and an encoder sequence can be used to establish an extended record tag from a series of binding events. In one embodiment, the first binding event uses a binding agent with a coding tag, and the coding tag comprises a universal 3' spacer primer sequence for the next binding cycle and an analyte-specific spacer sequence at the 5' end; the subsequent binding cycle then uses a binding agent with a coded analyte-specific 3' spacer sequence. This design results in the creation of amplifiable library elements only from a correct series of homologous binding events. The binding interactions of off-target and cross-reactions will result in non-amplifiable extended record tags. In one example, a pair of homologous binding agents of a specific macromolecular analyte is used in two binding cycles to identify the analyte. The first homologous binding agent contains a coding tag, which comprises a universal spacer 3' sequence for causing extension on the unive...

Claims

1. A composition comprising: (a) a solid support comprising at least 1,000,000 peptide molecules covalently bound to the solid support, wherein (i) each of the at least 1,000,000 peptide molecules is bound to a nucleic acid record tag, wherein each nucleic acid record tag bound to one of the at least 1,000,000 peptide molecules comprises a universal priming site for amplification and / or sequencing, and the at least 1,000,000 peptide molecules are randomly bound to the solid support; and (ii) adjacent peptide molecules on the solid support are spaced apart from each other by an average distance of greater than or equal to about 50 nm on the surface or within the volume of the solid support; and (b) a plurality of binding agents capable of binding to peptide molecules from said at least 1,000,000 peptide molecules, wherein each of said plurality of binding agents (i) comprises a nucleic acid encoding tag comprising an encoder sequence containing identification information about said binding agent, and (ii) binds to a feature or component of said peptide molecule.

2. A composition according to claim 1, wherein each of the peptide molecules is covalently bound to a corresponding nucleic acid record tag.

3. The composition according to claim 1, wherein each nucleic acid record tag is covalently bound to the C-terminal amino acid residue of the corresponding peptide molecule.

4. The composition of claim 1, wherein the solid support is a bead, a microsphere or a microparticle.

5. The composition of claim 1, wherein each nucleic acid record tag associated with a corresponding peptide molecule comprises a barcode or a unique molecular identifier (UMI).

6. The composition of claim 1, wherein each of the plurality of binding agents binds to an N-terminal amino acid residue, a C-terminal amino acid residue, a dipeptide sequence, a tripeptide sequence, a modified N-terminal amino acid residue, or a post-translationally modified amino acid residue of a peptide molecule of the at least 1,000,000 peptide molecules.

7. The composition of claim 1, wherein each peptide molecule covalently bound to the solid support comprises an N-terminal amino acid residue modified by a chemical reagent, and the modified N-terminal amino acid is bound by a cognate NTAA binding agent.

8. The composition of claim 1, wherein the binding agent is an aptamer, an antibody, an antibody binding fragment, a polypeptide or a protein.

9. The composition of claim 1, wherein each of the plurality of binding agents binds to an N-terminal amino acid residue or a C-terminal amino acid residue of a peptide molecule from the at least 1,000,000 peptide molecules.

10. The composition of claim 1, wherein the at least 1,000,000 peptide molecules covalently bound to the solid support are from different biological samples, and each associated nucleic acid tag comprises a sample-specific barcode.

11. The composition of claim 1, wherein the plurality of binding agents comprises at least three different binding agents.

12. A composition according to claim 1, wherein the nucleic acid encoding tag of at least one of the multiple binding agents binds to the nucleic acid recording tag associated with the peptide molecule from the at least 1,000,000 peptide molecules by hybridizing with a complementary sequence on the nucleic acid recording tag or forming a covalent bond with the nucleic acid recording tag.

13. The composition according to claim 1, wherein each of the nucleic acid recording tags comprises a spacer at its end, wherein the spacer has a sequence complementary to a sequence of a second spacer in the nucleic acid encoding tag.

14. The composition of claim 1, wherein each of the plurality of binding agents selectively binds to a feature or component of the peptide molecule.

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