Kit for analysis using nucleic acid coding and / or tags

Abstract

Kits and methods of analyzing macromolecules, including peptides, polypeptides, and proteins, using nucleic acid coding are disclosed. Sample analysis kits employ nucleic acid coding and / or nucleic acid recording of molecular interactions and / or reactions, such as recognition events (e.g., between antigens and antibodies, between modified terminal amino acid residues, or between small molecules or peptide therapeutic agents and targets, etc. Additional barcoding reagents, such as reagents for cycle-specific barcoding (e.g., "timing"), compartment barcoding reagents, combined barcoding reagents, spatial barcoding reagents, or any combination thereof, may be included in the kit. The sample may comprise macromolecules, including peptides, polypeptides, and proteins, and the recording may produce molecular interaction and / or reaction information, and / or polypeptide sequence information. The kits can be used for high throughput, multiplexing and / or automated analysis, and are suitable for the analysis of proteomes or subsets thereof.

Description

[0001] This case is a divisional application of a case with an application date of October 31, 2018, an invention title of "Kit for Analysis Using Nucleic Acid Coding and / or Labeling," and application number 201880084553.6.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to U.S. Provisional Application Serial No. 62 / 579,844, filed on October 31, 2017, entitled “Kits for performing assays using nucleic acid codes and / or tags,” and U.S. Provisional Application Serial No. 62 / 582,312, filed on November 6, 2017, entitled “Kits for performing assays using nucleic acid codes and / or tags,” and U.S. Provisional Application Serial No. 62 / 583,448, filed on November 8, 2017, entitled “Kits for performing assays using nucleic acid codes and / or tags,” the entire contents of each of which are incorporated herein by reference for all purposes. This application is related to U.S. Provisional Patent Application No. 62 / 330,841, filed May 2, 2016, entitled “Analysis of Macromolecules Using Nucleic Acid Encodings”; U.S. Provisional Patent Application No. 62 / 339,071, filed May 19, 2016, entitled “Analysis of Macromolecules Using Nucleic Acid Encodings”; U.S. Provisional Patent Application No. 62 / 376,886, filed August 18, 2016, entitled “Analysis of Macromolecules Using Nucleic Acid Encodings”; International Patent Application No. PCT / US2017 / 030702, filed May 2, 2017, entitled “Analysis of Macromolecules Using Nucleic Acid Encodings”; U.S. Provisional Patent Application No. 62 / 579,844, filed October 31, 2017, entitled “Kits for Performing Assays Using Nucleic Acid Encodings and / or Tags”, having attorney docket No. 776533000500; and U.S. Provisional Patent Application No. 62 / 579,844, filed October 31, 2017, entitled “Kits for Performing Assays Using Nucleic Acid Encodings and / or Tags”, having attorney docket No. 776533000500. U.S. Provisional Patent Application No. 62 / 579,870, entitled “Methods and Compositions for Polypeptide Analysis,” filed on October 31, 2017, having attorney docket No. 776533000700, entitled “Methods and Kits Using Nucleic Acid Encodings and / or Tags,” filed on November 6, 2017, having attorney docket No. 776533000501, entitled “Kits for Analysis Using Nucleic Acid Encodings and / or Tags,” and U.S. Provisional Patent Application No. 62 / 582,916, entitled “Methods and Kits Using Nucleic Acid Encodings and / or Tags,” filed on November 7, 2017, having attorney docket No. 776533000701, the disclosures of which are incorporated herein by reference for all purposes.

[0004] Submit sequence listings as ASCII text files

[0005] The following submission as an ASCII text file is incorporated herein by reference in its entirety: Computer Readable Form (CRF) of the Sequence Listing (File Name: 4614-2000540_20181031_SeqList.txt, Recording Date: October 31, 2018, Size: 52K Bytes). Technical Field

[0006] The present disclosure generally relates to sample analysis kits using nucleic acid encoding and / or nucleic acid recording of molecular interactions and / or reactions (e.g., recognition events). In some embodiments, the kits can be used for high-throughput, multiplexed and / or automated analysis and are suitable for analysis of a proteome or a subset thereof. Background Art

[0007] Proteins play an integral role in cell biology and physiology, performing and promoting many different biological functions. Due to the additional diversity introduced by post-translational modifications (PTMs), the collection of different protein molecules is very rich and much more complex than the transcriptome. 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 information related to genomic information. In general, innovation in proteomic analysis has lagged behind 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.

[0008] However, this protein information is urgently needed to better understand proteomic 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. The present disclosure addresses these and other needs. Summary of the Invention

[0009] This summary is not intended to be used to limit the scope of the claimed subject matter. Other features, details, utilities, and advantages of the claimed subject matter will become apparent from the detailed description, including those aspects disclosed in the accompanying drawings and the appended claims.

[0010] In any embodiment of the present disclosure, the coding tag and / or recording tag (and / or bitag, compartment tag, or partition tag, if applicable) or any portion thereof (e.g., universal primer, spacer, UMI, recording tag barcode, coding sequence, binding cycle-specific barcode, etc.) may include or be replaced by a sequenceable polymer, such as a non-nucleic acid sequenceable polymer.

[0011] In one aspect, a kit is disclosed herein comprising: (a) a recording tag configured to be directly or indirectly associated with an analyte; (b) (i) a coding tag containing identification information about a binding group capable of binding to the analyte and configured to be directly or indirectly associated with the binding group to form a binding agent, and / or (ii) a tag, wherein the recording tag and the coding tag are configured to allow information to be transferred between them upon binding between the binding agent and the analyte; and optionally, (c) a binding group. In one embodiment, the recording tag and / or the analyte are configured to be directly or indirectly fixed to a support. In another embodiment, the recording tag is configured to be fixed to a support, thereby fixing the analyte associated with the recording tag. In another embodiment, the analyte is configured to be fixed to a support, thereby fixing the recording tag associated with the analyte. In yet another embodiment, each of the recording tag and the analyte is configured to be fixed to a support. In another embodiment, the recording tag and the analyte are designed to be co-located when both are fixed to a support. In some embodiments, the distance between (i) the analyte and (ii) the recording tag for information transfer between the recording tag and the encoding tag of the binding agent bound to the analyte is less than about 10 -6 nm, about 10 -6 n, about 10 -5 nm, about 10 -4 nm, about 0.001 nm, about 0.01 nm, about 0.1 nm, about 0.5 nm, about 1 nm, about 2 nm, about 5 nm, or greater than about 5 nm, or any value within the foregoing range.

[0012] In any of the foregoing embodiments, the kit may further comprise an immobilization linker configured to: (i) be directly or indirectly immobilized on a support, and (ii) be directly or indirectly associated with the record tag and / or the analyte. In one embodiment, the immobilization linker is configured to be associated with the record tag and the analyte.

[0013] In any of the foregoing embodiments, the immobilization linker can be configured to be directly immobilized to the support, thereby immobilizing the record tag and / or analyte associated with the immobilization linker.

[0014] In any of the foregoing embodiments, the kit can further comprise a support.

[0015] In any of the foregoing embodiments, the kit may further comprise one or more reagents for transferring information between the coding tag and the recording tag upon binding between the binding agent and the analyte. In one embodiment, the one or more reagents are configured to transfer information from the coding tag to the recording tag, thereby generating an extended recording tag. In another embodiment, the one or more reagents are configured to transfer information from the recording tag to the coding tag, thereby generating an extended coding tag. In yet another embodiment, the one or more reagents are configured to generate a dual-tag construct comprising information from the coding tag and information from the recording tag.

[0016] In any of the foregoing embodiments, the test kit may include at least two record labels. In any of the foregoing embodiments, the test kit may include at least two coded labels, each coded label including identification information about its associated binding group. In a specific embodiment, each analyte has a plurality of record labels (e.g., at least about 2, about 5, about 10, about 20, about 50, about 100, about 200, about 500, about 1000, about 2000, about 5000 or more) that can be used to bind to the binding agent of the analyte. In a specific embodiment, the test kit includes a plurality of record labels, e.g., at least about 2, about 5, about 10, about 20, about 50, about 100, about 200, about 500, about 1000, about 2000, about 5000, or more.

[0017] In any of the foregoing embodiments, the kit may include at least two of the binding agents. In one embodiment, the kit includes: (i) one or more reagents for transferring information from a first coded tag of the first binding agent to a recording tag upon binding between the first binding agent and the analyte, thereby generating a first sequence-extended recording tag, and / or (ii) one or more reagents for transferring information from a coded tag of the second binding agent to the first extended recording tag upon binding between the second binding agent and the analyte, thereby generating a second sequence-extended recording tag, wherein the one or more reagents of (i) and the one or more reagents of (ii) may be the same or different. In specific embodiments, each analyte has multiple binding agents and / or coded tags, for example, at least about 2, about 5, about 10, about 20, about 50, about 100, about 200, about 500, about 1000, about 2000, about 5000, or more recording tags available for the analyte, and the multiple binding agents may be added sequentially or in parallel. In specific embodiments, the kit comprises a plurality of binding agents and / or coding tags, e.g., at least about 2, about 5, about 10, about 20, about 50, about 100, about 200, about 500, about 1000, about 2000, about 5000 or more.

[0018] In any of the foregoing embodiments, the kit may further comprise (iii) one or more reagents for transferring information from the third (or higher order) encoding label of the third (or higher order) binding agent to the second order extended recording label upon binding between the third (or higher order) binding agent and the analyte to produce a third (or higher order) order extended recording label. In one embodiment, the kit comprises: (i) one or more reagents for transferring information from a first coding tag of a first binding agent to a first recording tag upon binding between a first binding agent and an analyte to produce a first extended recording tag, (ii) one or more reagents for transferring information from a second coding tag of a second binding agent to a second recording tag upon binding between a second binding agent and an analyte to produce a second extended recording tag, and / or (iii) one or more reagents for transferring information from a third (or higher order) coding tag of a third (or higher order) binding agent to a third (or higher order) recording tag upon binding between a third (or higher order) binding agent and an analyte to produce a third (or higher order) extended recording tag, wherein one or more reagents of (ii), (iii) and / or (iii) may be the same or different.

[0019] In any of the foregoing embodiments, the kit may further comprise (iii) one or more reagents for transferring information from the third (or higher order) encoding label of the third (or higher order) binding agent to the third (or higher order) recording label upon binding between the third (or higher order) binding agent and the analyte to produce a third (or higher order) extended recording label.

[0020] In any of the foregoing embodiments, the first recording tag, the second recording tag, and / or the third (or higher order) recording tag may be configured to be directly or indirectly associated with the analyte.

[0021] In any of the foregoing embodiments, the first recording label, the second recording label, and / or the third (or higher order) recording label may be configured to be fixed on a support.

[0022] In any of the foregoing embodiments, the first recording tag, the second recording tag, and / or the third (or higher order) recording tag may be configured to be co-located with the analyte, for example, to allow transfer of information between the first, second, or third (or higher order) encoding tag and the first, second, or third (or higher order) recording tag, respectively, upon binding between the first, second, or third (or higher order) binding agent and the analyte.

[0023] In any of the foregoing embodiments, each of the first encoding tag, the second encoding tag, and / or the third (or higher order) encoding tag may comprise a bound cycle-specific barcode, such as a bound cycle-specific spacer sequence C nand / or encoding tag-specific spacer sequence C n , where n is an integer, and C n Indicates the binding between the nth binding agent and the polypeptide. Alternatively, a binding cycle tag C can be added exogenously n , for example, combined with the loop label C n The encoding tag may be of exogenous origin.

[0024] In any of the foregoing embodiments, the analyte can comprise a polypeptide. In one embodiment, the binding group of the kit is capable of binding to one or more N-terminal, internal, or C-terminal amino acids of the polypeptide, or is capable of binding to one or more N-terminal, internal, or C-terminal amino acids modified by a functionalizing agent.

[0025] In any of the foregoing embodiments, the kit can further comprise one or more functionalizing reagents.

[0026] In any of the foregoing embodiments, the kit may further comprise an elimination reagent for removing (e.g., by chemical or enzymatic cleavage) one or more N-terminal, internal or C-terminal amino acids of the polypeptide, or removing a functionalized N-terminal, internal or C-terminal amino acid, optionally wherein the elimination reagent comprises a carboxypeptidase or an aminopeptidase or a variant, mutant or modified protein thereof; a hydrolase or a variant, mutant or modified protein thereof; a mild Edman degradation reagent; an Edmanase enzyme; anhydrous TFA, a base; or any combination thereof.

[0027] In any of the foregoing embodiments, the one or more N-terminal, internal or C-terminal amino acids may comprise: (i) an N-terminal amino acid (NTAA); (ii) an N-terminal dipeptide sequence; (iii) an N-terminal tripeptide sequence; (iv) an internal amino acid; (v) an internal dipeptide sequence; (vi) an internal tripeptide sequence; (vii) a C-terminal amino acid; (viii) a C-terminal dipeptide sequence; or (ix) a C-terminal tripeptide sequence, or any combination thereof, optionally wherein any one or more amino acid residues in (i)-(ix) are modified or functionalized.

[0028] In another aspect, a kit is disclosed herein, comprising: at least (a) a first binding agent comprising (i) a first binding group capable of binding to the N-terminal amino acid (NTAA) or functionalized NTAA of a polypeptide to be analyzed, and (ii) a first coding tag comprising identification information about the first binding group, optionally (b) a recording tag configured to be directly or indirectly associated with the polypeptide, and further optionally (c) a functionalizing agent capable of modifying the first NTAA of the polypeptide to produce a first functionalized NTAA, wherein the recording tag and the first binding agent are configured to allow transfer of information between the first coding tag and the recording tag upon binding between the first binding agent and the polypeptide. In one embodiment, the kit further comprises one or more reagents for transferring information from the first coding tag to the recording tag, thereby producing a first sequence-extended recording tag.

[0029] In any of the foregoing embodiments, the functionalizing agent can comprise a chemical, enzyme, and / or biological agent, such as 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.

[0030] In any of the foregoing embodiments, the kit may further comprise an elimination reagent for removing (e.g., by chemical cleavage or enzymatic cleavage) the first functionalized NTAA to expose the immediately adjacent amino acid residue as a second NTAA. In one embodiment, the second NTAA is capable of being functionalized by the same or different functionalizing reagent to produce a second functionalized NTAA, which may be the same or different from the first functionalized NTAA. In another embodiment, the kit further comprises: (d) a second (or higher order) binding agent comprising (i) a second (or higher order) binding group capable of binding to the second functionalized NTAA, and (ii) a second (or higher order) coding tag comprising identification information about the second (or higher order) binding group, wherein the first coding tag and the second (or higher order) coding tag may be the same or different. In yet another embodiment, the first functionalized NTAA and the second functionalized NTAA are independently selected from the group consisting of a functionalized N-terminal alanine (A or Ala), cysteine ​​(C or Cys), aspartic acid (D or Asp), glutamate (E or Glu), phenylalanine (F or Phe), glycine (G or Gly), histidine (H or His), isoleucine (I or Ile), lysine (K or Lys), leucine (L or Leu), methionine (M or Met), asparagine (N or Asn), proline (P or Pro), glutamine (Q or Gln), arginine (R or Arg), serine (S or Ser), threonine (T or Thr), valine (V or Val), tryptophan (W or Trp), tyrosine (Y or Tyr), or any combination thereof.

[0031] In any of the foregoing embodiments, the kit may further comprise one or more reagents for transferring information from the second (or higher order) encoded label to the first order extended record label, thereby generating a second (or higher order) extended record label.

[0032] In another aspect, a kit is disclosed herein comprising: at least (a) one or more binding agents, each binding agent comprising (i) a binding group capable of binding to the N-terminal amino acid (NTAA) or functionalized NTAA of a polypeptide to be analyzed, and (ii) a coding tag comprising identification information about the binding group, and / or (b) one or more recording tags configured to be directly or indirectly associated with the polypeptide, wherein the one or more recording tags and the one or more binding agents are configured to allow information to be transferred between the coding tag and the recording tag upon binding between each binding agent and the polypeptide, and optionally (c) a functionalizing agent capable of modifying a first NTAA of the polypeptide to produce a first functionalized NTAA. In one embodiment, the kit further comprises an elimination reagent for removing (e.g., by chemical cleavage or enzymatic cleavage) the first functionalized NTAA to expose an adjacent amino acid residue (e.g., a second NTAA). In another embodiment, the second NTAA can be functionalized by the same or different functionalizing agent to produce a second functionalized NTAA, which can be the same or different from the first functionalized NTAA. In yet another embodiment, the first functionalized NTAA and the second functionalized NTAA are independently selected from the group consisting of a functionalized N-terminal alanine (A or Ala), cysteine ​​(C or Cys), aspartic acid (D or Asp), glutamate (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), valine (V or Val), tryptophan (W or Trp), tyrosine (Y or Tyr), or any combination thereof.

[0033] In any of the foregoing embodiments, the kit may comprise: (i) one or more reagents for transferring information from a first coding tag of a first binding agent to a first record tag upon binding between a first binding agent and a polypeptide, thereby producing a first extended record tag, and / or (ii) one or more reagents for transferring information from a second coding tag of a second binding agent to a second record tag upon binding between a second binding agent and a polypeptide, thereby producing a second extended record tag, wherein the one or more reagents of (i) and the one or more reagents of (ii) may be the same or different. In one aspect, the kit further comprises: (iii) one or more reagents for transferring information from a third (or higher order) coding tag of a third (or higher order) binding agent to a third (or higher order) record tag upon binding between a third (or higher order) binding agent and a polypeptide, thereby producing a third (or higher order) extended record tag.

[0034] In any of the foregoing embodiments, the first record label, the second record label, and / or the third (or higher order) record label may be configured to be directly or indirectly associated with the polypeptide.

[0035] In any of the foregoing embodiments, the first recording label, the second recording label, and / or the third (or higher order) recording label may be configured to be fixed on a support.

[0036] In any of the foregoing embodiments, the first recording tag, the second recording tag, and / or the third (or higher-order) recording tag can be configured to be co-localized with the polypeptide, for example, to allow transfer of information between the first, second, or third (or higher-order) encoding tag and the first, second, or third (or higher-order) recording tag, respectively, upon binding between the first, second, or third (or higher-order) binding agent and the polypeptide.

[0037] In any of the foregoing embodiments, the distance between the first recording label, the second recording label, and / or the third (or higher order) recording label on the support may be equal to or greater than about 10 nm, equal to or greater than about 15 nm, equal to or greater than about 20 nm, equal to or greater than about 50 nm, equal to or greater than about 100 nm, equal to or greater than about 150 nm, equal to or greater than about 200 nm, equal to or greater than about 250 nm, equal to or greater than about 300 nm, equal to or greater than about 350 nm, equal to or greater than about 400 nm, equal to or greater than about 450 nm, or equal to or greater than about 500 nm, and each recording label and its corresponding analyte are configured to co-localize when both are immobilized on the support, or the distance between each recording label and its corresponding analyte is less than about 10 nm. -6 nm, about 10 -6 nm, about 10 -5 nm, about 10 -4 nm, about 0.001 nm, about 0.01 nm, about 0.1 nm, about 0.5 nm, about 1 nm, about 2 nm, about 5 nm, or greater than about 5 nm, or any value therebetween.

[0038] In any of the foregoing embodiments, each of the first encoding tag, the second encoding tag, and / or the third (or higher order) encoding tag may comprise a bound cycle-specific barcode, such as a bound cycle-specific spacer sequence C n and / or encoding tag-specific spacer sequence C n , where n is an integer, and C n Indicates the binding between the nth binding agent and the polypeptide. Alternatively, a binding cycle tag C can be added exogenously n , for example, combined with the loop label C n The encoding tag may be of exogenous origin.

[0039] In any of the foregoing embodiments, the analyte or polypeptide can comprise a protein or polypeptide chain or fragment thereof, a lipid, a carbohydrate or a macrocycle, or a combination or complex thereof.

[0040] In any of the foregoing embodiments, the analyte or polypeptide can comprise a macromolecule or a complex thereof, such as a protein complex or a subunit thereof.

[0041] In any of the foregoing embodiments, the record label may comprise a nucleic acid, an oligonucleotide, a modified oligonucleotide, a DNA molecule, a DNA with pseudo-complementary bases, a DNA or RNA with one or more protected bases, an RNA molecule, a BNA molecule, an XNA molecule, an LNA molecule, a PNA molecule, a γPNA molecule, or a morpholino, or a combination thereof.

[0042] In any of the foregoing embodiments, the record label may include a universal priming site.

[0043] In any of the foregoing embodiments, the record tag may include a priming site for amplification, sequencing, or both, for example, the universal priming site includes a priming site for amplification, sequencing, or both.

[0044] In any of the foregoing embodiments, the recording label and / or the encoding label may include a unique molecular identifier (UMI).

[0045] In any of the foregoing embodiments, the record label and / or the encoding label may include a barcode and / or a nuclease site, such as a nicking endonuclease site (eg, a dsDNA nicking endonuclease site).

[0046] In any of the aforementioned embodiments, the recording tag and / or the encoding tag comprises a spacer at its 3'-end and / or at its 5'-end, for example, the recording tag comprises a spacer at its 3'-end.

[0047] In any of the foregoing embodiments, the record label and / or the coding label can include one or more nuclease sites, such as an endonuclease site, a homing endonuclease site, a restriction enzyme digestion site, a nicking endonuclease site, or a combination thereof. In certain embodiments, a nuclease site can be provided in the coding label, such as within the spacer sequence or between the spacer sequence and the encoder sequence. In certain embodiments, a nuclease site can be provided in the record label, such as between the universal primer sequence and the support (e.g., for cleaving the record label on the support).

[0048] In any of the foregoing embodiments, the kit can comprise a solid support, such as a rigid solid support, a flexible solid support, or a soft solid support, and including porous supports or non-porous supports.

[0049] In any of the foregoing embodiments, the kit can include a support comprising beads, porous beads, porous matrices, arrays, surfaces, glass surfaces, silicon surfaces, plastic surfaces, slides, filters, nylon, chips, silicon wafer chips, flow-through chips, biochips comprising signal transduction electronics, wells, microtiter wells, plates, ELISA plates, disks, rotating interferometry disks, membranes, nitrocellulose membranes, nitrocellulose-based polymer surfaces, nanoparticles (e.g., comprising metals such as magnetic nanoparticles (Fe3O4), gold nanoparticles, and / or silver nanoparticles), quantum dots, nanoshells, nanospheres, or any combination thereof. In one embodiment, the support comprises polystyrene beads, polymer beads, agarose beads, acrylamide beads, solid core beads, porous beads, paramagnetic beads, glass beads, or controlled pore beads, or any combination thereof.

[0050] In any of the foregoing embodiments, the kit can comprise a support and / or can be used to analyze multiple analytes (e.g., polypeptides) in sequential reactions, parallel reactions, or a combination of sequential and parallel reactions. In one embodiment, the analytes are spaced on the support at an average distance of about 10 nm or greater, about 15 nm or greater, about 20 nm or greater, about 50 nm or greater, about 100 nm or greater, about 150 nm or greater, about 200 nm or greater, about 250 nm or greater, about 300 nm or greater, about 350 nm or greater, about 400 nm or greater, about 450 nm or greater, or about 500 nm or greater.

[0051] In any of the foregoing embodiments, the binding group can comprise a polypeptide or fragment thereof, a protein or polypeptide chain or fragment thereof, or a protein complex or subunit thereof, such as an antibody or antigen-binding fragment thereof.

[0052] In any of the foregoing embodiments, the binding group may comprise a carboxypeptidase or an aminopeptidase, or a variant, mutant, or modified protein thereof; an aminoacyl-tRNA synthetase, or a variant, mutant, or modified protein thereof; avidin, or a variant, mutant, or modified protein thereof; ClpS, or a variant, mutant, or modified protein thereof; a UBR box protein, or a variant, mutant, or modified protein thereof; a modified small molecule that binds to an amino acid, i.e., vancomycin, or a variant, mutant, or modified molecule thereof; or any combination thereof, or wherein in each binding agent, the binding group comprises a small molecule and the encoding tag comprises a polynucleotide that recognizes the small molecule, whereby the plurality of binding agents form an encoded small molecule library, such as a DNA-encoded small molecule library.

[0053] In any of the foregoing embodiments, the binding group can selectively and / or specifically bind to the analyte or polypeptide.

[0054] In any of the foregoing embodiments, the encoding tag can comprise a nucleic acid, an oligonucleotide, a modified oligonucleotide, a DNA molecule, a DNA with pseudo-complementary bases, a DNA or RNA with one or more protected bases, an RNA molecule, a BNA molecule, an XNA molecule, an LNA molecule, a PNA molecule, a γPNA molecule, or a morpholino, or a combination thereof.

[0055] In any of the foregoing embodiments, the coding tag can comprise a barcode sequence, such as a coding region sequence, such as a coding sequence that recognizes a binding moiety.

[0056] In any of the foregoing embodiments, the encoded tag may comprise a spacer, a binding cycle-specific sequence, a unique molecular identifier (UMI), a universal priming site, or any combination thereof. In one embodiment, the binding cycle-specific sequence is added to the record tag after each binding cycle.

[0057] In any of the foregoing embodiments, the binding group and the coding tag can be connected via a linker or a binding pair.

[0058] In any of the foregoing embodiments, the binding group and the encoding tag can be connected by SpyTag / SpyCatcher, SpyTag-KTag / SpyLigase (wherein the two parts to be connected have a SpyTag / KTag pair and the SpyLigase connects the SpyTag to the KTag, thereby connecting the two parts), SnoopTag / SnoopCatcher peptide-protein pair, HaloTag / HaloTag ligand pair, or a sorting enzyme, such as LPXTG tag / sorting enzyme (e.g., Sortase A5, ActiveMotif, San Diego, or as disclosed in US 9,267,127 B2, which is incorporated herein by reference), or any combination thereof.

[0059] In any of the foregoing embodiments, the kit may further comprise a reagent for transferring information between the encoding tag and the recording tag in a templated or non-templated reaction, optionally wherein the reagent is (i) a chemical ligation reagent or a biological ligation reagent, for example, a ligase, such as a DNA ligase or RNA ligase for ligating single-stranded nucleic acids or double-stranded nucleic acids, or (ii) a reagent for extending single-stranded nucleic acid or double-stranded nucleic acid primers, optionally wherein the kit further comprises a ligation reagent, the ligation reagent comprising at least two ligases or variants thereof (e.g., at least two DNA ligases, or at least two RNA ligases, or at least one DNA ligase and at least one RNA ligase), wherein the at least two ligases or variants thereof comprise an adenylating ligase and a constitutively non-adenylated ligase, or optionally wherein the kit further comprises a ligation reagent, the ligation reagent comprising a DNA or RNA ligase and a DNA / RNA deadenylase.

[0060] In any of the foregoing embodiments, the kit may further comprise a polymerase, such as a DNA polymerase or an RNA polymerase or a reverse transcriptase, for transferring information between the encoding tag and the recording tag.

[0061] In any of the foregoing embodiments, the kit can further comprise one or more reagents for nucleic acid sequence analysis. In one embodiment, the nucleic acid sequence analysis comprises sequencing by synthesis, sequencing by ligation, sequencing by hybridization, polony sequencing, ion semiconductor sequencing, pyrosequencing, single molecule real-time sequencing, nanopore-based sequencing, or direct DNA imaging using advanced microscopy, or any combination thereof.

[0062] In any of the foregoing embodiments, the kit may further comprise one or more reagents for nucleic acid amplification, e.g., for amplifying one or more extended record tags, optionally wherein the nucleic acid amplification comprises an exponential amplification reaction (e.g., polymerase chain reaction (PCR), e.g., emulsion PCR to reduce or eliminate template switching) and / or a linear amplification reaction (e.g., isothermal amplification by in vitro transcription, or isothermal chimeric primer-initiated nucleic acid amplification (ICAN)). See, for example, Uemori et al. (2007), “Investigation of themolecular mechanism of ICAN, a novel gene amplification method,” J Biochem 142(2): 283-292; Mukai et al. (2007), “Highly efficient isothermal DNA amplification system using three elements of 5'-DNA-RNA-3' chimeric primers, RNaseH and strand-displacing DNA polymerase,” J Biochem 142(2): 273-281; ​​Ma et al. (2013), “Isothermal amplification method for next-generation sequencing,” Proc Natl Acad Sci US A. 110(35):14320–14323. 110(35):14320-14323, the entirety of which is incorporated herein by reference for all purposes.

[0063] In any of the foregoing embodiments, the kit may comprise one or more reagents for transferring the encoded tag information to the record tag to form an extended record tag, wherein the order and / or frequency of the encoded tag information on the extended record tag indicates the order and / or frequency of binding of the binding agent to the analyte or polypeptide.

[0064] In any of the foregoing embodiments, the kit can further comprise one or more reagents for target enrichment, for example, enrichment of one or more extended record tags.

[0065] In any of the foregoing embodiments, the kit can further comprise one or more reagents for subtraction, such as subtraction of one or more extended record tags.

[0066] In any of the foregoing embodiments, the kit can further comprise one or more reagents for normalization, eg, to reduce high abundance species, such as one or more analytes or polypeptides.

[0067] In any of the foregoing embodiments, at least one binding agent of the kit can bind to the terminal amino acid residue, the terminal two amino acid residues, or the terminal three amino acid residues.

[0068] In any of the preceding embodiments, at least one binding agent of the kit can bind to a post-translationally modified amino acid.

[0069] In any of the foregoing embodiments, the test kit may further include one or more reagents or devices for partitioning the multiple analytes or polypeptides in the sample into multiple compartments, wherein each compartment includes multiple compartment labels, and multiple compartment labels are optionally connected to a support (e.g., a solid support), wherein the multiple compartment labels are identical within a single compartment and are different from the compartment labels of other compartments. In one embodiment, the test kit also includes one or more reagents or tools for fragmenting multiple analytes or polypeptides (e.g., multiple protein complexes, proteins and / or polypeptides) into multiple polypeptide fragments.

[0070] In any of the foregoing embodiments, the kit can further comprise one or more reagents or means for annealing or ligating the plurality of polypeptide fragments to the compartment tags within each of the plurality of compartments, thereby generating a plurality of compartment-tagged polypeptide fragments.

[0071] In any of the foregoing embodiments, the plurality of compartments can comprise microfluidic droplets, microwells, or separate areas on a surface, or any combination thereof.

[0072] In any of the foregoing embodiments, each of the plurality of compartments may comprise, on average, a single cell.

[0073] In any of the foregoing embodiments, the kit can further comprise one or more universal DNA tags for labeling multiple analytes or polypeptides in the sample.

[0074] In any of the foregoing embodiments, the kit can further comprise one or more reagents for labeling a plurality of analytes or polypeptides in a sample with one or more universal DNA tags.

[0075] In any of the foregoing embodiments, the kit can further comprise one or more reagents for primer extension or ligation.

[0076] In any of the foregoing embodiments, the support can comprise polystyrene beads, polymer beads, agarose beads, acrylamide beads, solid core beads, porous beads, paramagnetic beads, glass beads, or controlled pore beads, or any combination thereof.

[0077] In any of the foregoing embodiments, the compartment tag can comprise a single-stranded or double-stranded nucleic acid molecule.

[0078] In any of the foregoing embodiments, the compartment labels can comprise a barcode and, optionally, a UMI. In any of the foregoing embodiments, the support can be a bead, and the compartment labels can comprise a barcode.

[0079] In any of the foregoing embodiments, the support can comprise beads, and the beads comprising the plurality of compartment tags attached thereto can be formed by split and pool synthesis, separate synthesis, or immobilization, or any combination thereof.

[0080] In any of the foregoing embodiments, the kit can further comprise one or more reagents for split and pooled synthesis, separate synthesis, or immobilization, or any combination thereof.

[0081] In any of the foregoing embodiments, the compartment tag can be a component within a record tag, wherein the record tag optionally further comprises a spacer, a barcode sequence, a unique molecular identifier, a universal priming site, or any combination thereof.

[0082] In any of the foregoing embodiments, the compartment tag can further comprise a functional moiety capable of reacting with an internal amino acid, a peptide backbone, or an N-terminal amino acid on a plurality of analytes or polypeptides (e.g., a protein complex, a protein, or a polypeptide). In one embodiment, the functional moiety can comprise an aldehyde, an azide / alkyne, a maleimide / thiol, an epoxide / nucleophile, an inverse electron demand Diels-Alder (iEDDA) group, a click reagent, or any combination thereof.

[0083] In any of the foregoing embodiments, the compartment tag can further comprise a peptide, such as a protein ligase recognition sequence, and optionally the protein ligase can be butelase I or a homolog thereof.

[0084] In any of the foregoing embodiments, the kit can further comprise a chemical or biological reagent, such as an enzyme, such as a protease (eg, a multi-metalloprotease), for fragmenting the plurality of analytes or polypeptides.

[0085] In any of the foregoing embodiments, the kit can further comprise one or more reagents for releasing the compartment tags from the support.

[0086] In any of the foregoing embodiments, the kit may further comprise one or more reagents for forming an extended encoding tag or ditag construct. In one embodiment, the 3' end of the recording tag is blocked to prevent extension of the recording tag by a polymerase. In any of the foregoing embodiments, the encoding tag may comprise an encoder sequence, a UMI, a universal priming site, a spacer at its 3' end, a binding cycle-specific sequence, or any combination thereof.

[0087] In any of the foregoing embodiments, the ditag construct can be generated by gap filling, primer extension, or a combination thereof.

[0088] In any of the above embodiments, the di-tag molecule comprises a universal priming site derived from a recording tag, a compartment tag derived from a recording tag, a compartment tag derived from a recording tag, a unique molecular identifier derived from a recording tag, an optional spacer derived from a recording tag, a coding sequence derived from an encoding tag, a unique molecular identifier derived from an encoding tag, an optional spacer derived from an encoding tag, and a universal priming site derived from an encoding tag.

[0089] In any of the foregoing embodiments, the binding agent can be a polypeptide or a protein.

[0090] In any of the foregoing embodiments, the binding agent may comprise an aminopeptidase or a variant, mutant, or modified protein thereof; an aminoacyl-tRNA synthetase or a variant, mutant, or modified protein thereof; avidin or a variant, mutant, or modified protein thereof; ClpS or a variant, mutant, or modified protein thereof; or a modified small molecule that binds to an amino acid, i.e., vancomycin or a variant, mutant, or modified molecule thereof (e.g., vancomycin that binds to D-alanyl-D-alanine); or an antibody or binding fragment thereof; or any combination thereof.

[0091] In any of the foregoing embodiments, the binding agent can bind to a single amino acid residue (e.g., an N-terminal amino acid residue, a C-terminal amino acid residue, or an internal amino acid residue), a dipeptide (e.g., an N-terminal dipeptide, a C-terminal dipeptide, or an internal dipeptide), a tripeptide (e.g., an N-terminal tripeptide, a C-terminal tripeptide, or an internal tripeptide), or a post-translationally modified analyte or polypeptide.

[0092] In any of the foregoing embodiments, the binding agent can bind to the N-terminal polypeptide, the C-terminal polypeptide, or an internal polypeptide.

[0093] In any of the foregoing embodiments, the encoding tag and / or recording tag may comprise one or more error correction codes, one or more encoder sequences, one or more barcodes, one or more UMIs, one or more compartment labels, one or more cycle-specific sequences, or any combination thereof. In some embodiments, the error correction code is selected from the group consisting of: a Hamming code, a Lie distance code, an asymmetric Lie distance code, a Reed-Solomon code, and a Levenshtein-Tenengolts code.

[0094] In any of the foregoing embodiments, the encoding tag and / or the recording tag may include a loop tag.

[0095] In any of the foregoing embodiments, the kit can further comprise a circulation tag independent of the encoding tag and / or the recording tag.

[0096] In any of the foregoing embodiments, the kit may further comprise: (a) reagents for producing cell lysates or protein samples; (b) reagents for blocking amino acid side chains, such as by alkylation of cysteine ​​or blocked lysine; (c) proteases, such as trypsin, LysN, or LysC; (d) reagents for immobilizing nucleic acid-labeled polypeptides (e.g., DNA-labeled proteins) to a support; (e) reagents for degradation-based polypeptide sequencing; and / or (f) reagents for nucleic acid sequencing.

[0097] In any of the foregoing embodiments, the kit may comprise: (a) reagents for producing a cell lysate or protein sample; (b) reagents for blocking amino acid side chains, such as by alkylation of cysteine ​​or blocking lysine; (c) a protease, such as trypsin, LysN or LysC; (d) reagents for immobilizing a polypeptide (e.g., a protein) to a support comprising an immobilized recording label; (e) reagents for degradation-based polypeptide sequencing; and / or (f) reagents for nucleic acid sequencing.

[0098] In any of the foregoing embodiments, the kit can include: (a) reagents for producing a cell lysate or protein sample; (b) denaturing reagents; (c) reagents for blocking amino acid side chains, such as by alkylation of cysteine ​​or blocked lysine; (d) universal DNA primer sequences; (e) reagents for labeling polypeptides with universal DNA primer sequences; (f) barcoded beads for annealing the labeled polypeptides with primers; (g) reagents for polymerase extension, for writing the barcode from the beads to the labeled polypeptides; (h) a protease, such as trypsin, LysN or LysC; (i) reagents for immobilizing nucleic acid-labeled polypeptides (e.g., DNA-labeled proteins) to a support; (j) reagents for degradation-based polypeptide sequencing; and / or (k) reagents for nucleic acid sequencing.

[0099] In any of the foregoing embodiments, the kit can include: (a) a cross-linking agent; (b) reagents for generating a cell lysate or protein sample; (c) reagents for blocking amino acid side chains, such as by alkylation of cysteine ​​or blocking lysine; (d) a universal DNA primer sequence; (e) reagents for labeling polypeptides with the universal DNA primer sequence; (f) barcoded beads for annealing the labeled polypeptides with primers; (g) reagents for polymerase extension, for writing the barcode from the beads to the labeled polypeptides; (h) a protease, such as trypsin, LysN or LysC; (i) reagents for immobilizing nucleic acid-labeled polypeptides (e.g., DNA-labeled proteins) to a support; (j) reagents for degradation-based polypeptide sequencing; and / or (k) reagents for nucleic acid sequencing.

[0100] In any of the foregoing embodiments of the kit, one or more components can be provided in solution or on a support, such as a solid support.

[0101] Kit components may also include any molecule, molecular complex or conjugate, reagent (e.g., chemical or biological reagent), agent, structure (e.g., support, surface, particle or bead), reaction intermediate, reaction product, binding complex, or any other article disclosed and / or used in the following exemplary methods and / or aspects. The present kit can be used to analyze any suitable analyte, e.g., a macromolecule or polypeptide. In some embodiments, the kits of the present invention can be used for highly parallel, high-throughput digital analysis (e.g., small molecule analysis), particularly polypeptide analysis. In some embodiments, the kits of the present invention can be used in the following exemplary methods for analyzing an analyte, e.g., a macromolecule or polypeptide.

[0102] In the first example, there is a method for analyzing an analyte, such as a macromolecule or polypeptide, comprising the following steps: (a) providing an analyte and a recording label connected to a solid support; (b) contacting the analyte with a first binding agent capable of binding to the analyte, wherein the first binding agent comprises a first coding label having identification information about the first binding agent; (c) transferring information from the first coding label to the recording label to produce a first-order extended recording label; (d) contacting the analyte with a second binding agent capable of binding to the analyte, wherein the second binding agent comprises a second coding label having identification information about the second binding agent; (e) transferring information from the second coding label to the first-order extended recording label to produce a second-order extended recording label; and (f) analyzing the second-order extended label.

[0103] In a second example is the method of the first example, wherein contacting steps (b) and (d) are performed sequentially.

[0104] In a third example is the method of the first example wherein contacting steps (b) and (d) are performed simultaneously.

[0105] In the fourth example, the method according to the first example 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 modified analyte, wherein the third (or higher order) binding agent contains 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).

[0106] In a fifth example, there is a method for analyzing an analyte, such as a macromolecule or polypeptide, comprising the following steps: (a) providing an analyte, an associated first recording label, and an associated second recording label attached to a solid support; (b) contacting the analyte with a first binding agent capable of binding to the analyte, wherein the first binding agent comprises a first coded label having identification information about the first binding agent; (c) transferring information from the first coded label to the recording label to produce a first-order extended recording label; (d) contacting the analyte with a second binding agent capable of binding to the analyte, wherein the second binding agent comprises a second coded label having identification information about the second binding agent; (e) transferring information from the second coded label to the second recording label to produce a second-order extended recording label; and (f) analyzing the first and second extended recording labels.

[0107] In a sixth example, the method of the fifth example is described, wherein the contacting steps (b) and (d) are performed sequentially.

[0108] In a seventh example, the method of the fifth example is described, wherein the contacting steps (b) and (d) are performed simultaneously.

[0109] In an eighth example, the method of the fifth example is described, wherein step (a) further comprises providing an associated third (or higher order) recording label attached to the solid support.

[0110] In the ninth example, the method according to the eighth example is further provided, further comprising 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 modified analyte, 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 a third (or higher order) recording tag to produce a third (or higher order) extended recording tag; and analyzing the first, second and third (or higher order) extended recording tags in step (f).

[0111] In a tenth example is the method of any one of the fifth to ninth examples, wherein the first encoding tag, the second encoding tag, and any higher order encoding tags comprise a bound cycle-specific spacer sequence.

[0112] In the 11th example, there is a method for analyzing a peptide, comprising the steps of: (a) providing a peptide and an associated recording tag connected to a solid support; (b) modifying the N-terminal amino acid (NTAA) of the peptide with a chemical agent; (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 recording tag to produce an extended recording tag; and (e) analyzing the extended recording tag.

[0113] In the 12th example, the method according to the 11th example is described, wherein step (c) further includes contacting the peptide with a second (or higher order) binding agent comprising a second (or higher order) coding tag, the 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 a modified NTAA other than the modified NTAA of step (b).

[0114] In the 19th embodiment, the method of the 18th embodiment is described, wherein after the peptide is contacted with the first binding agent, the peptide is then contacted with a second (or higher order) binding agent.

[0115] In a 14th embodiment, the method of the 12th embodiment is provided, wherein contacting the peptide with the second (or higher order) binding agent occurs simultaneously with contacting the peptide with the first binding agent.

[0116] In the 15th example, it is a method according to any one of examples 11 to 14, 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.

[0117] In Example 21, a method for analyzing a peptide comprises 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 agent 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 about the first binding agent; (d) transferring the information of the first coding tag to the record tag to produce a first extended record tag; (e) removing the modified NTAA to expose a new NTAA; (f) modifying the new NTAA of the peptide with a chemical agent 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 tag having identification information about the second binding agent; h) transferring the information of the second coding tag to the first extended record tag to produce a second extended record tag; and (i) analyzing the second extended record tag.

[0118] In Example 21, a method for analyzing a peptide comprises the following steps: (a) providing a peptide and an associated recording 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 recording tag to produce an extended recording tag; and (d) analyzing the extended recording tag.

[0119] In the 18th example, the method according to the 17th example is provided, wherein step (b) further comprises contacting the peptide with a second (or higher order) binding agent comprising a second (or higher order) coding tag, the 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.

[0120] In the 19th embodiment, the method of the 18th embodiment is described, wherein after the peptide is contacted with the first binding agent, the peptide is then contacted with a second (or higher order) binding agent.

[0121] In the 20th embodiment, the method of the 18th embodiment is provided, wherein contacting the peptide with the second (or higher order) binding agent occurs simultaneously with contacting the peptide with the first binding agent.

[0122] In Example 21, a method for analyzing a peptide comprises the following steps: (a) providing a peptide and an associated recording 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 information from the first coding tag to the recording tag to produce a first extended recording tag; (d) removing the NTAA to expose a new NTAA of the peptide; (e) contacting the peptide with a second binding agent capable of binding to the new NTAA, wherein the second binding agent comprises a second coding tag having identification information about the second binding agent; (f) transferring information from the second coding tag to the first extended recording tag to produce a second extended recording tag; and (g) analyzing the second extended recording tag.

[0123] In a twenty-second example, the method according to any one of the first to tenth examples is provided, wherein the analyte is a protein, a polypeptide, or a peptide.

[0124] In a 23rd example, the method according to any one of the 1st to 10th examples is provided, wherein the analyte is a peptide.

[0125] In a 24th example, the method according to any one of examples 11 to 23, wherein the peptide is obtained by fragmenting a protein isolated from a biological sample.

[0126] In a twenty-fifth example, the method of any one of the first to tenth examples is provided, wherein the analyte is a lipid, a carbohydrate, or a macrocycle.

[0127] In the 26th example, the method according to any one of the first to 25th examples is provided, wherein the recording tag is a DNA molecule, a DNA having pseudo-complementary bases, an RNA molecule, a BNA molecule, an XNA molecule, an LNA molecule, a PNA molecule, a γPNA molecule, or a combination thereof.

[0128] In a 27th example, the method according to any one of the first to 26th examples is described, wherein the record label includes a universal priming site.

[0129] In the 28th example, the method of the 27th example is described, wherein the universal priming site comprises a priming site for amplification, sequencing, or both.

[0130] In a 29th example, the method of any one of the first to 28th examples is described, wherein the record tag includes a unique molecular identifier (UMI).

[0131] In a 30th example, the method according to any one of the first to 29th examples is provided, wherein the record label includes a barcode.

[0132] In a 31st example, the method according to any one of the first to 30th examples is described, wherein the record tag includes a spacer at its 3'-end.

[0133] In a 32nd example, the method according to any one of the 1st to 31st examples is provided, wherein the analyte and the associated recording tag are covalently attached to a solid support.

[0134] In the 33rd example, a method according to any one of the first to 32nd examples is provided, 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.

[0135] In the 34th example, the method of the 33rd example is described, 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.

[0136] In a thirty-fifth example, is a method according to any one of the first to thirty-fourth examples, wherein a plurality of analytes (eg, molecules of the same analyte or different analytes) and associated recording tags are attached to a solid support.

[0137] In a 36th example, is the method of the 35th example, wherein the plurality of analytes (eg, molecules of the same analyte or different analytes) are spaced apart on the solid support at an average distance of >50 nm.

[0138] In the 37th example, the method of any one of the 1st to 36th examples is described, wherein the binding agent is a polypeptide or protein.

[0139] In the 38th example, the method of the 37th example is described, wherein the binding agent is a modified aminopeptidase, a modified aminoacyl-tRNA synthetase, a modified anticalin, or a modified ClpS.

[0140] In a 39th example, the method of any one of the 1st to 38th examples is provided, wherein the binding agent is capable of selectively binding the analyte.

[0141] In a 40th example, the method according to any one of the first to 39th examples is described, wherein the recording label 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.

[0142] In a 41st example, the method of any one of the first to 40th examples is described, wherein the encoding tag comprises an encoder sequence.

[0143] In a 42nd example, the method according to any one of the first to 41st examples is provided, 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.

[0144] In a 43rd example, the method according to any one of the first to 42nd examples is described, wherein the binding agent and the coding tag are connected by a linker.

[0145] In the 44th example, a method according to any one of the first to 42nd examples is described, wherein the binder and the coding tag are connected by SpyTag / SpyCatcher, SpyTag-KTag / SpyLigase (wherein the two parts to be connected have a SpyTag / KTag pair, and SpyLigase connects the SpyTag to the KTag, thereby connecting the two parts), a sorting enzyme, or a SnoopTag / SnoopCatcher peptide-protein pair.

[0146] In a 45th example, the method according to any one of the 1st to 44th examples is described, wherein the information transfer from the encoding tag to the recording tag is mediated by DNA ligase.

[0147] In a 46th example, the method according to any one of the first to 44th examples is described, wherein the information transfer from the encoding tag to the recording tag is mediated by DNA polymerase.

[0148] In a 47th example, the method according to any one of the first to 44th examples is described, wherein the transfer of information from the encoding tag to the recording tag is mediated by chemical linkage.

[0149] In a 48th example, the method according to any one of the first to 47th examples is described, wherein analyzing the extended record tag comprises a nucleic acid sequencing method.

[0150] In the 49th example, the method according to the 48th example, wherein the nucleic acid sequencing method is by synthesis sequencing, by ligation sequencing, by hybridization sequencing, polony sequencing, ion semiconductor sequencing or pyrophosphate sequencing.

[0151] In the 50th example, a method according to the 48th example is provided, wherein the nucleic acid sequencing method is single-molecule real-time sequencing, nanopore-based sequencing, or direct DNA imaging using an advanced microscope.

[0152] In a 51st example, the method according to any one of the first to 50th examples is described, wherein the extended recording tag is amplified before analysis.

[0153] In a 52nd example, is the method according to any one of the 1st to 51st examples, wherein the order of the encoded tag information contained on the extended recording tags provides information about the binding order of the binding agents that bind to the analyte.

[0154] In a fifty-third example, is a method according to any one of the first to fifty-second examples, wherein the frequency of the encoded tag information contained on the extended recording tag provides information about the binding frequency of the binding agent bound to the analyte.

[0155] In a 54th example, a method according to any one of the 1st to 53rd examples is described, wherein a plurality of extended recording tags representing a plurality of analytes (eg, molecules of the same analyte or different analytes) are analyzed in parallel.

[0156] In a fifty-fifth example, is a method according to the fifty-fourth example, wherein a plurality of extended recording tags representing multiple analytes (eg, molecules of the same analyte or different analytes) are analyzed in a multiplexed assay.

[0157] In a 56th example, the method of any one of the 1st to 55th examples is described, wherein the plurality of extended record tags are subjected to a target enrichment assay prior to analysis.

[0158] In a fifty-seventh example, the method of any one of the first to fifty-sixth examples is described, wherein the plurality of extended recording tags are subjected to a subtraction assay prior to analysis.

[0159] In a 58th example, the method according to any one of the first to 57th examples is described, wherein the plurality of extended recording tags are normalized prior to analysis to reduce high abundance species.

[0160] In the 59th example, the method of any one of the 1st to 58th examples is described, wherein NTAA is removed using a modified aminopeptidase, a modified amino acid tRNA synthetase, mild Edman degradation, Edmanase enzyme, or anhydrous TFA.

[0161] In the 60th example, the method according to any one of the 1st to 59th examples is provided, wherein at least one binding agent binds to a terminal amino acid residue.

[0162] In the 61st example, is a method according to any one of the first to 60th examples, wherein at least one binding agent binds to a post-translationally modified amino acid.

[0163] In Example 67, a method for analyzing one or more peptides from a sample comprising multiple protein complexes, proteins or polypeptides is provided, the method comprising: (a) distributing the multiple protein complexes, proteins or polypeptides within the sample into multiple compartments, wherein each compartment comprises multiple compartment tags optionally attached to a solid support, wherein the multiple compartment tags are the same in a single compartment and different from the compartment tags of other compartments; (b) fragmenting the multiple protein complexes, proteins and / or polypeptides into multiple peptides; (c) contacting the multiple peptides with the multiple compartment tags under conditions sufficient to allow the multiple peptides to anneal or attach to the multiple compartment tags in the multiple compartments, thereby producing multiple compartment-labeled peptides; (d) collecting the compartment-labeled peptides from the multiple compartments; and (e) analyzing the one or more compartment-labeled peptides according to the method described in any one of Examples 1 to 21 and Examples 26 to 61.

[0164] In Example 63, the method of Example 62 is described, wherein the compartment is a microfluidic droplet.

[0165] In a 64th example, the method of the 62nd example is described, wherein the compartment is a microwell.

[0166] In a 65th example, a method according to the 62nd example, wherein the compartments are separate areas on the surface.

[0167] In Example 66, the method of any one of Examples 62 to 65, wherein each compartment contains an average of one individual cell.

[0168] In Example 67, a method for analyzing one or more peptides from a sample comprising multiple protein complexes, proteins, or polypeptides is described, the method comprising: (a) labeling multiple protein complexes, proteins, or polypeptides with multiple universal DNA tags; (b) distributing the multiple labeled protein complexes, proteins, or polypeptides within the sample into multiple compartments, wherein each compartment comprises multiple compartment tags, wherein the multiple compartment tags are the same in a single compartment and different from the compartment tags of other compartments; (c) contacting the multiple protein complexes, proteins, or polypeptides with multiple compartment tags under conditions sufficient to allow the multiple protein complexes, proteins, or polypeptides to anneal or connect to the multiple compartment tags in the multiple compartments, thereby producing multiple compartment-labeled protein complexes, proteins, or polypeptides; (d) collecting the compartment-labeled protein complexes, proteins, or polypeptides from the multiple compartments; (e) optionally fragmenting the compartment-labeled protein complexes, proteins, or polypeptides into compartment-labeled peptides; and (f) analyzing the one or more compartment-labeled peptides according to the method described in Examples 1 to 21 and any one of Examples 26 to 61.

[0169] In a 68th example, the method according to any one of examples 62 to 67, wherein the compartment label information is transferred to a record tag associated with the peptide by primer extension or ligation.

[0170] In the 69th example, the method according to any one of examples 62 to 68 is described, wherein the solid support comprises beads.

[0171] In the 70th example, the method of the 69th example is described, 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.

[0172] In the 71st example, the method according to any one of the 62nd to 70th examples is described, wherein the compartment tag comprises a single-stranded or double-stranded nucleic acid molecule.

[0173] In the 72nd example, the method of any one of the 62nd to 71st examples is provided, wherein the compartment labels include a barcode and optionally a UMI.

[0174] In the 73rd example, the method according to the 72nd example, wherein the solid support is a bead, the compartment label comprises a barcode, and further wherein the bead comprising a plurality of compartment labels attached thereto is synthesized by equalization and merging.

[0175] In the 74th example, the method according to the 72nd example, 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.

[0176] In a 75th example, the method of any one of examples 62 to 74 is provided, wherein the compartment tag is a component of a record tag, the record tag optionally further comprising a spacer, a unique molecular identifier, a universal priming site, or any combination thereof.

[0177] In the 76th example, the method according to any one of examples 62 to 75, wherein the compartment tag further comprises a functional portion capable of reacting with an internal amino acid or an N-terminal amino acid on the plurality of protein complexes, proteins or polypeptides.

[0178] In the 77th example, the method of the 76th example is provided, wherein the functional moiety is an NHS group.

[0179] In the 78th example, there is provided a method according to the 76th example, wherein the functional moiety is an aldehyde group.

[0180] In a 79th example, the method of any one of examples 62 to 78 is provided, wherein the plurality of compartment labels are formed by printing, spotting, inkjet printing the compartment labels into the compartments, or a combination thereof.

[0181] In the 80th example, the method according to any one of the 62nd to 79th examples is described, wherein the compartment tag further comprises a peptide.

[0182] In the 81st example, the method according to any one of the 80th examples, wherein the compartment tag peptide comprises a protein ligase recognition sequence.

[0183] In the 82nd example, the method of the 81st example is described, wherein the protein ligase is butelase I or a homolog thereof.

[0184] In the 83rd example, the method according to any one of examples 62 to 82, wherein the plurality of polypeptides are fragmented using a protease.

[0185] In an 84th example, the method of the 83rd example is described, wherein the protease is a metalloprotease.

[0186] In the 85th example, the method of the 84th example is described, wherein the activity of the metalloprotease is regulated by photon-stimulated release of metal cations.

[0187] In an 86th example, the method of any one of examples 62 to 85 further comprises subtracting one or more abundant proteins from the sample before partitioning the plurality of polypeptides into the plurality of compartments.

[0188] In the 87th example, the method according to any one of examples 62 to 86, further comprising releasing the compartment tags from the solid support before attaching the plurality of peptides to the compartment tags.

[0189] In Example 88, the method according to Example 62 further comprises the following step (d), linking the compartment tag peptide to a solid support associated with the record tag.

[0190] In the 89th example, the method according to the 88th example further comprises transferring information of the compartment label on the compartment-tagged peptide to the associated record label.

[0191] In the 90th example, the method of the 89th example further comprises removing the compartment tag from the compartment-labeled peptide before step (e).

[0192] In the 91st example, the method according to any one of examples 62 to 90 further comprises determining the identity of the single cell from which the analyzed peptide originated based on the compartment tag sequence of the analyzed peptide.

[0193] In the 92nd example, the method according to any one of examples 62 to 90 further comprises determining the identity of the source protein or protein complex of the analyzed peptide based on the compartment tag sequence of the analyzed peptide.

[0194] In Example 93, a method for analyzing multiple analytes (e.g., molecules of the same analyte or different analytes) is provided, the method comprising the following steps: (a) providing multiple analytes and associated recording tags attached to a solid support; (b) contacting the multiple analytes with multiple binding agents capable of binding to the multiple analytes, wherein each binding agent comprises a coding tag having identification information about the binding agent; (c) (i) transferring the information of the recording tag associated with the analyte to the coding tag of the binding agent bound to the analyte to produce an extended coding tag; or (ii) transferring the information of the recording tag associated with the analyte and the coding tag of the binding agent bound to the analyte to a dual-tag construct; (d) collecting the extended coding tags or dual-tag constructs; (e) optionally repeating steps (b)-(d) for one or more binding cycles; and (f) analyzing the collection of extended coding tags or dual-tag constructs.

[0195] In a 94th example, the method of the 93rd example is provided, wherein the analyte is a protein.

[0196] In the 95th example, a method according to the 93rd example is provided, wherein the analyte is a peptide.

[0197] In the 96th example, the method of the 95th example is provided, wherein the peptide is obtained by fragmenting a protein isolated from a biological sample.

[0198] In a 97th example, the method according to any one of the 93rd to 96th examples is described, wherein the recording 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.

[0199] In a 98th example, the method of any one of the 93rd to 97th implementations is provided, wherein the record tag comprises a unique molecular identifier (UMI).

[0200] In a 99th example, the method according to any one of the 93rd to 98th examples is provided, wherein the record label includes a compartment label.

[0201] In a 100th example, the method according to any one of the 93rd to 99th examples is provided, wherein the record label includes a universal priming site.

[0202] In a 101st example, the method according to any one of the 93rd to 100th examples is described, wherein the record tag includes a spacer at its 3'-end.

[0203] In the 102nd example, a method according to any one of examples 93 to 101 is described, wherein the 3'-end of the recording tag is blocked to prevent extension of the recording tag by a synthetase, and information of the analyte-associated recording tag and information of the encoding tag of the binding agent bound to the analyte are transferred to a dual-tag construct.

[0204] In Example 103, the method of any one of Examples 93 to 102, wherein the coding tag comprises a coding sequence.

[0205] In the 104th example, the method of any one of the 93rd to 103rd examples is provided, wherein the encoded tag comprises a UMI.

[0206] In the 105th example, the method according to any one of the 93rd to 104th examples is described, wherein the encoding tag comprises a universal priming site.

[0207] In the 106th example, according to any one of the 93rd to 105th examples, the coding tag comprises a spacer at its 3'-end.

[0208] In the 107th example, the method according to any one of the 93rd to 106th examples is described, wherein the encoding tag comprises a binding cycle-specific sequence.

[0209] In the 108th example, the method according to any one of the 93rd to 107th examples is described, wherein the binding agent and the coding tag are connected by a linker.

[0210] In a 109th example, the method according to any one of the 93rd to 108th examples is described, wherein the information transfer from the recording tag to the encoding tag is achieved by primer extension.

[0211] In a 110th example, the method according to any one of the 93rd to 108th examples is provided, wherein the information transfer from the recording tag to the encoding tag is achieved by connection.

[0212] In the 111th example, the method of any one of examples 93 to 108, wherein the ditag construct is generated by gap filling, primer extension, or both.

[0213] In the 112th example, a method according to any one of examples 93-97, 107, 108 and 111 is described, wherein the dual-tag molecule comprises a universal priming site derived from a recording tag, a compartment tag derived from a recording tag, a unique molecular identifier derived from a recording tag, an optional spacer derived from a recording tag, a coding sequence derived from a coding tag, a unique molecular identifier derived from a coding tag, an optional spacer derived from a coding tag and a universal priming site derived from a coding tag.

[0214] In a 113th example, the method of any one of examples 93 to 112, wherein the analyte and the associated recording tag are covalently bound to a solid support.

[0215] In the 114th example, the method according to the 113th example, 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.

[0216] In Example 115, the method of Example 114 is described, 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.

[0217] In the 116th example, the method according to any one of examples 93 to 115, wherein the binding agent is a polypeptide or protein.

[0218] In Example 117, the method of Example 116 is described, wherein the binding agent is a modified aminopeptidase, a modified aminoacyl-tRNA synthetase, a modified anticalin, or an antibody or a binding fragment thereof.

[0219] In the 118th example is a method according to any one of examples 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.

[0220] In the 119th example, the method according to the 118th example is described, 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.

[0221] In example 120, the method of example 118, wherein the binding agent binds to the N-terminal peptide, the C-terminal peptide, or an internal peptide.

[0222] In the 121st example, the method of example 119, wherein the binding agent binds to the N-terminal amino acid residue and the N-terminal amino acid residue is cleaved after each binding cycle.

[0223] In example 122, the method of example 119, wherein the binding agent binds to the C-terminal amino acid residue and cleaves the C-terminal amino acid residue after each binding cycle.

[0224] Example 123. The method of Example 121, wherein the N-terminal amino acid residue is cleaved by Edman degradation.

[0225] Example 124. The method of Example 93, wherein the binding agent is a site-specific covalent tag of an amino acid or a post-translational modification.

[0226] Example 125. The method of any one of Examples 93-124, wherein after step (b), the complex comprising the analyte and the associated binding agent is dissociated from the solid support and dispersed into droplets or a microfluidic droplet emulsion.

[0227] Example 126. The method of Example 125, wherein each microfluidic droplet contains, on average, a complex comprising an analyte and a binding agent.

[0228] Example 127. The method of example 125 or 126, wherein the record tags are amplified prior to generating the extended encoding tags or di-tag constructs.

[0229] Example 128. The method of any one of Examples 125 to 127, wherein emulsion fusion PCR is used to transfer the record tag information to the encoding tag or to create a set of dual-tag constructs.

[0230] Example 129. The method of any one of Examples 93 to 128, wherein the collection of extension-encoding tags or di-tag constructs is amplified prior to analysis.

[0231] Example 130. The method of any one of Examples 93 to 129, wherein analyzing the collection of extended-encoded tags or di-tag constructs comprises a nucleic acid sequencing method.

[0232] Example 131. The method of Example 130, wherein the nucleic acid sequencing method is sequencing by synthesis, sequencing by ligation, sequencing by hybridization, polony sequencing, ion semiconductor sequencing, or pyrophosphate sequencing.

[0233] Example 132. A method according to Example 130, wherein the nucleic acid sequencing method is single molecule real-time sequencing, nanopore-based sequencing, or direct imaging of DNA using advanced microscopy.

[0234] Example 133. The method of example 130, wherein the partial composition of the analyte is determined by analyzing a plurality of extended encoding tags or dual tagging tag constructs using specific compartment tags or, alternatively, using UMIs.

[0235] Example 134. A method according to any one of Examples 1 to 133, wherein the analyzing step is performed using a sequencing method with a per base error rate of >5%, >10%, >15%, >20%, >25% or >30%.

[0236] Example 135. A method according to any one of Examples 1 to 134, wherein the identification element of the encoding label, the recording label, or both includes an error correction code.

[0237] Example 136. The method of Example 135, wherein the identification element is selected from an encoder sequence, a barcode, a UMI, a compartment tag, a cycle-specific sequence, or any combination thereof.

[0238] Example 137. The method of Example 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.

[0239] Example 138. A method according to any one of Examples 1 to 134, wherein the identification element 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 analyzing step comprises detecting the unique current or ion flux or optical signature to identify the identification element.

[0240] Example 139. The method of Example 138, wherein the identification element is selected from an encoder sequence, a barcode, a UMI, a compartment tag, a cycle-specific sequence, or any combination thereof.

[0241] Example 140. A method for analyzing a plurality of analytes (e.g., molecules of the same analyte or different analytes), the method comprising the following steps: (a) providing a plurality of analytes and associated record labels attached to a solid support; (b) contacting the plurality of analytes with a plurality of binding agents capable of binding to homologous analytes, wherein each binding agent comprises a coding label having identification information about the binding agent; (c) transferring information from a first coding label of a first binding agent to a first record label associated with a first analyte to produce a first sequence-extended record label, wherein the first binding agent binds to the first sequence-extended record label. analyte; (d) contacting the plurality of analytes with a plurality of binding agents capable of binding to the homologous analyte; (e) transferring the information of the second coding tag of the second binding agent to the first order extended record tag to produce a second order extended record tag, wherein the second binding agent is bound to the first analyte; (f) optionally repeating steps (d)-(e) for "n" binding cycles, wherein the information of each coding tag of each binding agent bound to the first analyte is transferred to the extended record tag produced in the previous binding cycle to produce an nth order extended record tag representing the first analyte; (g) analyzing the nth order extended record tag.

[0242] Example 141. The method of Example 140, wherein a plurality of n-th order extended record tags representing a plurality of analytes are generated and analyzed.

[0243] Example 142. The method of Example 140 or 141, wherein the analyte is a protein.

[0244] Example 143. The method of Example 142, wherein the analyte is a peptide.

[0245] Example 144. The method of Example 143, wherein the peptides are obtained by fragmenting proteins from a biological sample.

[0246] Example 145. A method according to any one of Examples 140 to 144, wherein the plurality of analytes comprises analytes (e.g., macromolecules such as polypeptides, proteins, protein complexes) from a plurality of pooled samples.

[0247] Example 146. The method of any one of Examples 140 to 145, wherein the record tag 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.

[0248] Example 147. The method of any one of Examples 140 to 146, wherein the record tag comprises a unique molecular identifier (UMI).

[0249] Example 148. The method of any one of Examples 140 to 147, wherein the record label comprises a compartment label.

[0250] Example 149. The method of any one of Examples 140 to 148, wherein the record label comprises a universal priming site.

[0251] Example 150. The method of any one of Examples 140 to 149, wherein the record tag comprises a spacer at its 3'-end.

[0252] Example 151. A method according to any one of Examples 140 to 150, wherein the encoding tag comprises an encoder sequence.

[0253] Example 152. The method of any one of Examples 140 to 151, wherein the encoded tag comprises a UMI.

[0254] Example 153. The method of any one of Examples 140 to 152, wherein the encoding tag comprises a universal priming site.

[0255] Example 154. The method of any one of Examples 140 to 153, wherein the encoding tag comprises a spacer at its 3'-end.

[0256] Example 155. A method according to any one of Examples 140 to 154, wherein the encoding tag comprises a binding cycle-specific sequence.

[0257] Example 156. A method according to any one of Examples 140 to 155, wherein the encoding tag comprises a unique molecular identifier.

[0258] Example 157. The method of any one of Examples 140 to 156, wherein the binding agent and the coding tag are connected by a linker.

[0259] Example 158. The method of any one of Examples 140 to 157, wherein the information transfer from the recording tag to the encoding tag is mediated by primer extension.

[0260] Example 159. A method according to any one of Examples 140 to 158, wherein the transfer of information from the recording tag to the encoding tag is mediated by a connection.

[0261] Example 160. The method of any one of Examples 140 to 159, wherein the plurality of analytes, associated recording tags, or both are covalently bound to a solid support.

[0262] Example 161. According to any one of the methods described in Examples 140 to 160, 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.

[0263] Example 162. The method of Example 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.

[0264] Example 163. The method of any one of Examples 140 to 162, wherein the binding agent is a polypeptide or protein.

[0265] Example 164. The method of Example 163, wherein the binding agent is a modified aminopeptidase, a modified aminoacyl-tRNA synthetase, a modified anticalin, or an antibody or binding fragment thereof.

[0266] Example 165. The method of any one of Examples 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.

[0267] Example 166. The method of Example 165, wherein the binding agent binds to an N-terminal amino acid residue, a C-terminal amino acid residue, or an internal amino acid residue.

[0268] Example 167. The method of Example 165, wherein the binding agent binds to the N-terminal peptide, the C-terminal peptide, or an internal peptide.

[0269] Example 168. The method of any one of Examples 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.

[0270] Example 169. A method according to Example 166 or 168, wherein the binding agent binds to a chemical tag at the N-terminal amino acid residue or a modified N-terminal amino acid residue, and the N-terminal amino acid residue is cleaved after each binding cycle.

[0271] Example 170. A method according to Example 166 or 168, wherein the binding agent binds to a chemical tag of the C-terminal amino acid residue or a modified C-terminal amino acid residue, and the C-terminal amino acid residue is cleaved after each binding cycle.

[0272] Example 171. The method of Example 169, wherein the N-terminal amino acid residue is cleaved by Edman degradation, Edmanase, a modified aminopeptidase, or a modified acyl peptide hydrolase.

[0273] Example 172. The method of Example 163, wherein the binding agent is a site-specific covalent tag of an amino acid or a post-translational modification.

[0274] Example 173. The method of any one of Examples 140 to 172, wherein the plurality of n-th order extended record tags are amplified prior to analysis.

[0275] Example 174. The method of any one of Examples 140 to 173, wherein analyzing the n-th order extended record tag comprises a nucleic acid sequencing method.

[0276] Example 175. The method of Example 174, wherein multiple n-th order extended record tags representing multiple analytes are analyzed in parallel.

[0277] Example 176. The method of Example 174 or 175, wherein the nucleic acid sequencing method is sequencing by synthesis, sequencing by ligation, sequencing by hybridization, polony sequencing, ion semiconductor sequencing, or pyrophosphate sequencing.

[0278] Example 177. A method according to Example 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.

[0279] Example 1'. A method comprising: (a) contacting a set of proteins with a library of agents, wherein each protein is directly or indirectly associated with a record tag, wherein each agent comprises (i) a small molecule, a peptide or peptide mimetic, a peptide (e.g., a peptoid, a β-peptide, or a D-peptide peptidomimetic), a polysaccharide, or an aptamer (e.g., a nucleic acid aptamer, such as a DNA aptamer or a peptide aptamer), and (ii) a coding tag comprising a recognition tag for the small molecule, peptide or peptide mimetic, peptide (e.g., a peptoid, a β-peptide, or a D-peptide peptidomimetic), polysaccharide, or aptamer. (b) allowing the transfer of information between (i) the record tag associated with each protein and / or its associated record tag, or each reagent, directly or indirectly immobilized on a support, and (ii) the coding tag of one or more reagents to generate an extended record tag and / or an extended coding tag; and (c) analyzing the extended record tag and / or the extended coding tag.

[0280] Example 2'. The method of Example 1', wherein each protein is spaced apart from other proteins on the support by an average distance equal to or greater than about 20 nm, equal to or greater than about 50 nm, equal to or greater than about 100 nm, equal to or greater than about 150 nm, equal to or greater than about 200 nm, equal to or greater than about 250 nm, equal to or greater than about 300 nm, equal to or greater than about 350 nm, equal to or greater than about 450 nm, equal to or greater than about 500 nm, equal to or greater than about 550 nm, equal to or greater than about 600 nm, equal to or greater than about 650 nm, equal to or greater than about 700 nm, equal to or greater than about 750 nm, equal to or greater than about 800 nm, equal to or greater than about 850 nm, equal to or greater than about 900 nm, equal to or greater than about 950 nm, or equal to or greater than about 1 μm.

[0281] Example 3'. The method of Example 1' or 2', wherein each protein and its associated record label is spaced apart from other proteins and their associated record labels on the support by an average distance equal to or greater than about 20 nm, equal to or greater than about 50 nm, equal to or greater than about 100 nm, equal to or greater than about 150 nm, equal to or greater than about 200 nm, equal to or greater than about 250 nm, equal to or greater than about 300 nm, equal to or greater than about 350 nm, equal to or greater than about 400 nm, equal to or greater than about 450 nm, equal to or greater than about 500 nm, equal to or greater than about 550 nm, equal to or greater than about 600 nm, equal to or greater than about 650 nm, equal to or greater than about 700 nm, equal to or greater than about 750 nm, equal to or greater than about 800 nm, equal to or greater than about 850 nm, equal to or greater than about 900 nm, equal to or greater than about 950 nm, or equal to or greater than about 1 μm.

[0282] Example 4'. A method according to any one of Examples 1' to 3', wherein one or more proteins and / or their associated record tags are covalently fixed to the support (e.g., via a linker) or non-covalently fixed to the support (e.g., via a binding pair).

[0283] Example 5'. The method according to any one of Examples 1' to 4', wherein a subset of the proteins and / or their associated record labels are covalently immobilized on the support, and another subset of the proteins and / or their associated record labels are non-covalently immobilized on the support.

[0284] Example 6'. The method according to any one of Examples 1' to 5', wherein one or more recording tags are fixed to a support, thereby fixing the relevant protein.

[0285] Example 7'. The method according to any one of Examples 1' to 6', wherein one or more proteins are immobilized on a support, thereby immobilizing the associated record label.

[0286] Example 8'. The method according to any one of Examples 1 to 7, wherein at least one protein is co-localized with its associated recording tag while each is independently immobilized on a support.

[0287] Example 9'. A method according to any one of Examples 1' to 8', wherein at least one protein and / or its associated record label is directly or indirectly associated with a fixed linker, and the fixed linker is directly or indirectly fixed to a support, thereby fixing at least one protein and / or its associated record label to the support.

[0288] Example 10′. The method according to any one of Examples 1′ to 9′, wherein the density of the immobilized recording tags is equal to or greater than the density of the immobilized proteins.

[0289] Example 11'. The method of Example 10', wherein the density of the immobilized recording labels is at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 20 times, at least about 50 times, at least about 100 times, or more, the density of the immobilized proteins.

[0290] Example 12'. A method according to Example 1', wherein the average distance between each reagent and the other reagents immobilized on the support is equal to or greater than about 20 nm, equal to or greater than about 50 nm, equal to or greater than about 100 nm, equal to or greater than about 150 nm, equal to or greater than about 200 nm, equal to or greater than about 250 nm, equal to or greater than about 300 nm, equal to or greater than about 350 nm, equal to or greater than about 400 nm, equal to or greater than about 450 nm, equal to or greater than about 500 nm, equal to or greater than about 550 nm, equal to or greater than about 600 nm, equal to or greater than about 650 nm, equal to or greater than about 700 nm, equal to or greater than about 750 nm, equal to or greater than about 800 nm, equal to or greater than about 850 nm, equal to or greater than about 900 nm, equal to or greater than about 950 nm, or equal to or greater than about 1 μm.

[0291] Example 13'. The method of Example 12', wherein one or more reagents are covalently immobilized to the support (eg, via a linker), or non-covalently immobilized to the support (eg, via a binding pair).

[0292] Example 14'. The method of example 12' or 13', wherein a subset of the reagents are covalently immobilized to the support and another subset of the reagents are non-covalently immobilized to the support.

[0293] Example 15'. A method according to any one of Examples 12' to 14', wherein for one or more reagents, a small molecule, a peptide or peptide mimetic, a peptide (e.g., a peptoid, a β-peptide or a D-peptide peptide mimetic), a polysaccharide or an aptamer is fixed to a support to fix the encoding tag.

[0294] Example 16'. A method according to any one of Examples 12' to 15', wherein for one or more reagents, a coding tag is fixed to a support to fix a small molecule, a peptide or peptide mimetic, a peptide (e.g., a peptoid, a β-peptide, or a D-peptide peptide mimetic), a polysaccharide, or an aptamer.

[0295] Example 17'. The method of any of Examples 1' to 16', wherein information is transferred from at least one encoding label to at least one recording label, thereby producing at least one extended recording label.

[0296] Example 18'. The method of any of Examples 1' to 17', wherein information is transferred from at least one recording label to at least one encoding label, thereby producing at least one extended encoding label.

[0297] Example 19'. The method of any one of Examples 1' to 18', wherein at least one di-tag construct is generated, the di-tag construct comprising information from the encoding tag and information from the recording tag.

[0298] Example 20'. A method according to any one of Examples 1' to 19', wherein at least one protein is bound and / or reacted with two or more agents selected from small molecules, peptides or peptide mimetics, peptides (e.g., peptoids, β-peptides or D-peptide peptide mimetics), polysaccharides or aptamers.

[0299] Example 21'. A method according to Example 20', wherein the extended recording label or extended coding label contains identification information about two or more agents of small molecules, peptides or peptide mimetics, peptides (e.g., peptoids, β-peptides or D-peptide peptide mimetics), polysaccharides or aptamers.

[0300] Example 22'. The method of any one of Examples 1' to 21', wherein at least one of the proteins is associated with two or more record labels, wherein the two or more record labels can be the same or different.

[0301] Example 23'. The method of any one of Examples 1' to 22', wherein at least one of the reagents comprises two or more coding tags, wherein the two or more coding tags can be the same or different.

[0302] Example 24'. A method according to any one of Examples 1' to 23', wherein the transfer of information is accomplished by ligation (e.g., enzymatic or chemical ligation, splint ligation, sticky-end ligation, single-stranded (ss) ligation such as ssDNA ligation, or any combination thereof), a polymerase-mediated reaction (e.g., primer extension of single-stranded nucleic acid or double-stranded nucleic acid), or any combination thereof.

[0303] Example 25'. A method according to Example 24', wherein the ligation and / or polymerase-mediated reaction has faster kinetics relative to the binding occupancy time or reaction time between the protein and the small molecule, peptide or peptide mimetic, peptide (e.g., peptoid, β-peptide, or D-peptide peptide mimetic), polysaccharide, or aptamer, optionally, wherein the reagents for the ligation and / or polymerase-mediated reaction are present in the same reaction volume as the binding or reaction between the protein and the small molecule, peptide or peptide mimetic, peptide (e.g., peptoid, β-peptide or D-peptide peptide mimetic, polysaccharide or aptamer), and further optionally wherein information transfer is achieved by slowing the dissociation rate of the binding agent using a concomitant binding / encoding step, and / or by using a reduced temperature for the encoding or information writing step.

[0304] Example 26'. A method according to any one of Examples 1' to 25', wherein each protein is associated with its recording tag by a separate attachment, and / or wherein each small molecule, peptide or peptide mimetic, peptide (e.g., a peptoid, β-peptide, or D-peptide peptide mimetic), polysaccharide or aptamer is associated with its encoding tag by a separate attachment.

[0305] Example 27'. The method of Example 26', wherein attachment occurs via ribosome or mRNA / cDNA presentation, wherein the recording tag and / or encoding tag sequence information is contained in the mRNA sequence.

[0306] Example 28'. The method according to Example 27', wherein the recording tag and / or the encoding tag comprises a universal primer sequence, a barcode and / or a spacer sequence at the 3' end of the mRNA sequence.

[0307] Example 29'. The method according to Example 28', wherein the recording tag and / or the encoding tag further comprises a restriction enzyme digestion site at the 3' end.

[0308] Example 30'. A method according to any one of Examples 1' to 29', wherein the set of proteins is a proteome or a subset thereof, optionally wherein the set of proteins is produced using in vitro transcription of a genome or a subset thereof followed by in vitro translation, or produced using in vitro translation of a transcriptome or a subset thereof.

[0309] Example 31'. A method according to Example 30', wherein the subset of the proteome includes a kinome; a secretome; a receptorome (e.g., a GPCRome); an immune proteome; a nutritional proteome; a subset of the proteome defined by post-translational modification (e.g., phosphorylation, ubiquitination, methylation, acetylation, glycosylation, oxidation, lipidation and / or nitrosylation), such as a phosphoproteomic (e.g., a phosphotyrosine-proteome, a tyrosine-kinase group and a tyrosine-phosphatase group), a glycoproteome, etc.; a subset of the proteome associated with a tissue or organ, a developmental stage or a physiological or pathological condition; a subset of the proteome associated with a cellular process such as the cell cycle, differentiation (or dedifferentiation), cell death, aging, cell migration, transformation or metastasis; or any combination thereof.

[0310] Example 32'. A method according to any one of Examples 1' to 31', wherein the group of proteins is from a mammal, such as a human, a non-human animal, a fish, an invertebrate, an arthropod, an insect or a plant, such as yeast, a bacterium, such as Escherichia coli, a virus, such as HIV or HCV, or a combination thereof.

[0311] Example 33'. The method according to any one of Examples 1' to 32', wherein the group of proteins comprises a protein complex or a subunit thereof.

[0312] Example 34'. A method according to any one of Examples 1' to 33', wherein the record label comprises a nucleic acid, an oligonucleotide, a modified oligonucleotide, a DNA molecule, a DNA with pseudo-complementary bases, an RNA molecule, a BNA molecule, an XNA molecule, an LNA molecule, a PNA molecule, a γPNA molecule or a morpholino, or a combination thereof.

[0313] Example 35'. The method of any one of Examples 1' to 34', wherein the record label comprises a universal priming site.

[0314] Example 36'. The method of any one of Examples 1' to 35', wherein the record tag comprises a priming site for amplification, sequencing, or both, eg, the universal priming site comprises a priming site for amplification, sequencing, or both.

[0315] Example 37'. The method of any one of Examples 1' to 36', wherein the record tag comprises a unique molecular identifier (UMI).

[0316] Example 38'. The method of any one of Examples 1' to 37', wherein the record label comprises a barcode.

[0317] Example 39'. The method of any one of Examples 1-38, wherein the record tag comprises a spacer at its 3'-end.

[0318] Example 40'. The method according to any one of Examples 1-39, wherein the support is a solid support, such as a rigid solid support, a flexible solid support, or a soft solid support, and includes a porous support or a non-porous support.

[0319] Example 41'. A method according to any one of Examples 1' to 40', wherein the support comprises beads, porous beads, magnetic beads, paramagnetic beads, a porous matrix, an array, a surface, a glass surface, a silicon surface, a plastic surface, a slide, a filter, nylon, a chip, a silicon crystal chip, a flow chip, a biochip including a signal transduction electronic device, a well, a microtiter well, a plate, an ELISA plate, a disk, a rotating interferometry disk, a membrane, a nitrocellulose membrane, a nitrocellulose-based polymer surface, nanoparticles (e.g., containing metals such as magnetic nanoparticles (Fe3O4), gold nanoparticles and / or silver nanoparticles), quantum dots, nanoshells, nanospheres, or any combination thereof.

[0320] Example 42'. The method of Example 41', wherein the support comprises polystyrene beads, polymer beads, agarose beads, acrylamide beads, solid core beads, porous beads, magnetic beads, paramagnetic beads, glass beads or controlled pore beads, or any combination thereof.

[0321] Example 43'. A method according to any one of Examples 1' to 42', which is used to analyze in parallel the interactions between the group of proteins and small molecule libraries, and / or peptides or peptide mimetics, and / or peptides (e.g., peptoids, β-peptides, or D-peptide peptidomimetics), and / or polysaccharides, and / or aptamers to produce a small molecule-protein binding matrix, and / or a peptide / peptide mimetic-protein binding matrix, and / or a peptide-protein binding matrix (e.g., a peptoid-protein binding matrix, a β-peptide-protein binding matrix, or a D-peptide mimetic-protein binding matrix), and / or a polysaccharide-protein binding matrix, and / or an aptamer-protein binding matrix.

[0322] Example 44'. The method of Example 43', wherein the matrix size is about 10 2 , about 10 3 , about 10 4 , about 10 5 , about 10 6 , about 10 7 , about 10 8 , about 10 9 , about 10 10 , about 10 11 , about 10 12 , about 10 13 , about 10 14 or larger, such as about 2×10 13 .

[0323] Example 45'. A method according to any one of Examples 1' to 44', wherein the encoding tag comprises a nucleic acid, an oligonucleotide, a modified oligonucleotide, a DNA molecule, a DNA with pseudo-complementary bases, an RNA molecule, a BNA molecule, an XNA molecule, an LNA molecule, a PNA molecule, a γPNA molecule or a morpholino, or a combination thereof.

[0324] Example 46'. The method of any one of Examples 1' to 45', wherein the encoding tag comprises an encoder sequence that recognizes a small molecule, a peptide or peptide mimetic, a peptide (e.g., a peptoid, a β-peptide or a D-peptide peptide mimetic), a polysaccharide or an aptamer.

[0325] Example 47'. The method of any one of Examples 1' to 46', wherein the encoding tag comprises a spacer, a unique molecular identifier (UMI), a universal priming site, or any combination thereof.

[0326] Example 48'. A method according to any one of Examples 1' to 47', wherein the small molecule, peptide or peptide mimetic, peptide (e.g., peptoid, β-peptide, or D-peptide peptide mimetic), polysaccharide, or aptamer and the encoding tag are connected via a linker or binding pair.

[0327] Example 49'. A method according to any one of Examples 1' to 48', wherein the small molecule, peptide or peptide mimetic, peptide class (e.g., peptoid, β-peptide or D-peptide peptide mimetic), polysaccharide or aptamer and encoding tag are connected by SpyTag-KTag / SpyLigase (wherein the two parts to be connected have a SpyTag-KTag / SpyLigase pair and SpyLigase connects the SpyTag to KTag, thereby connecting the two parts), SpyTag / SpyCatcher, SnoopTag / SnoopCatcher peptide-protein pair, sortase, or HaloTag / HaloTag ligand pair, or any combination thereof.

[0328] Example 50'. A method for analyzing a polypeptide, comprising: (a) contacting (i) a group of fragments of a polypeptide with (ii) a library of binding agents, wherein each fragment is directly or indirectly associated with a record tag, wherein each binding agent comprises a binding group and a coding tag, the coding tag comprising identification information about the binding group, wherein the binding group is capable of binding to one or more N-terminal, internal or C-terminal amino acids of the fragment, or is capable of binding to one or more N-terminal, internal or C-terminal amino acids modified by a functionalizing agent, and wherein each fragment and / or its associated record tag, or each binding agent is directly or indirectly fixed to a support; (b) when the binding group binds to one or more N-terminal, internal or C-terminal amino acids of the fragment, allowing the transfer of information between (i) the record tag associated with each fragment and (b) the coding tag to produce an extended record tag and / or an extended coding tag; and (c) analyzing the extended record tag and / or the extended coding tag.

[0329] Example 51'. A method according to Example 50', wherein one or more N-terminal, internal or C-terminal amino acids comprise: (i) an N-terminal amino acid (NTAA); (ii) an N-terminal dipeptide sequence; (iii) an N-terminal tripeptide sequence; (iv) an internal amino acid; (v) an internal dipeptide sequence; (vi) an internal tripeptide sequence; (vii) a C-terminal amino acid; (viii) a C-terminal dipeptide sequence; or (ix) a C-terminal tripeptide sequence, or any combination thereof, optionally wherein any one or more amino acid residues in (i)-(ix) are modified or functionalized.

[0330] Example 52'. The method of Example 51', wherein one or more N-terminal, internal or C-terminal amino acids are independently selected at each residue from the group consisting of: alanine (A or Ala), cysteine ​​(C or Cys), aspartic acid (D or Asp), glutamate (E or Glu), phenylalanine (F or Phe), glycine (G or Gly), histidine (H or His), isoleucine (I or Ile), lysine (K or Lys), leucine (L or Leu), methionine (M or Met), asparagine (N or Asn), proline (P or Pro), glutamine (Q or Gln), arginine (R or Arg), serine (S or Ser), threonine (T or Thr), valine (V or Val), tryptophan (W or Trp), tyrosine (Y or Tyr), or any combination thereof.

[0331] Example 53'. A method according to any one of Examples 50' to 52', wherein the binding group comprises a polypeptide or a fragment thereof, a protein or a polypeptide chain or a fragment thereof, or a protein complex or a subunit thereof, such as an antibody or an antigen-binding fragment thereof.

[0332] Example 54'. A method according to any one of Examples 50' to 53', wherein the binding group comprises an antibody or a variant, mutant or modified protein thereof; an aminoacyl-tRNA synthetase or a variant, mutant or modified protein thereof; avidin or a variant, mutant or modified protein thereof; ClpS or a variant, mutant or modified protein thereof; a UBR box protein or a variant, mutant or modified protein thereof; or a modified small molecule that binds to an amino acid, i.e., vancomycin or a variant, mutant or modified molecule thereof; or any combination thereof.

[0333] Example 55'. A method according to any one of Examples 50' to 54', wherein the binding group is capable of selectively and / or specifically binding to a functionalized N-terminal amino acid (NTAA), an N-terminal dipeptide sequence or an N-terminal tripeptide sequence or any combination thereof.

[0334] Example 56'. A method for analyzing a plurality of polypeptides, comprising: (a) labeling each molecule of the plurality of polypeptides with a plurality of universal tags; (b) contacting the plurality of polypeptides with a plurality of compartment tags under conditions suitable for annealing or ligation of the plurality of universal tags to the plurality of compartment tags, thereby partitioning the plurality of polypeptides into a plurality of compartments (e.g., a bead surface, a microfluidic droplet, a microwell, or separate areas on a surface, or any combination thereof), wherein the plurality of compartment tags are identical within each compartment and different from the compartment tags of other compartments; (c) fragmenting the polypeptides in each compartment, thereby generating a set of polypeptide fragments, each polypeptide fragment being associated with a record tag comprising at least one universal polynucleotide tag and at least one compartment tag; and (d) directly or indirectly immobilized on a support; (e) contacting the immobilized polypeptide fragment group with a library of binding agents, wherein each binding agent comprises a binding group and a coding tag, the coding tag comprising identification information about the binding group, wherein the binding group is capable of binding to one or more N-terminal, internal or C-terminal amino acids of the fragment, or is capable of binding to one or more N-terminal, internal, or C-(c)-terminal amino acids modified by a functionalizing agent; (f) when the binding group binds to one or more N-terminal, internal or C-terminal amino acids of the fragment, allowing the transfer of information between (i) the record tag associated with each fragment and (ii) the coding tag to produce an extended record tag and / or an extended coding tag; (g) resolving the extended record tag and / or the extended coding tag.

[0335] Example 57'. The method of Example 56', wherein the multiple polypeptides having the same compartment tag belong to the same protein.

[0336] Example 58'. The method of Example 56', wherein the multiple polypeptides having the same compartment tag belong to different proteins, for example, two, three, four, five, six, seven, eight, nine, ten or more proteins.

[0337] Example 59'. The method of any of Examples 56' to 58', wherein a plurality of compartment labels are secured to a plurality of substrates, each substrate defining a compartment.

[0338] Example 60'. A method according to Example 59', wherein the plurality of substrates are selected from the group consisting of beads, porous beads, magnetic beads, paramagnetic beads, porous matrices, arrays, surfaces, glass surfaces, silicon surfaces, plastic surfaces, slides, filters, nylon, chips, silicon crystal chips, flow chips, biochips comprising signal transduction electronic devices, wells, microtiter wells, plates, ELISA plates, disks, rotating interferometry disks, membranes, nitrocellulose membranes, nitrocellulose-based polymer surfaces, nanoparticles (e.g., comprising metals such as magnetic nanoparticles (Fe3O4), gold nanoparticles and / or silver nanoparticles), quantum dots, nanoshells, nanospheres, or any combination thereof.

[0339] Example 61'. A method according to Example 59' or 60', wherein each of the multiple substrates comprises barcoded particles, such as barcoded beads, for example, polystyrene beads, polymer beads, agarose beads, acrylamide beads, solid core beads, porous beads, magnetic beads, paramagnetic beads, glass beads or controlled pore beads, or any combination thereof.

[0340] Example 62'. A method according to any one of Examples 59'-61', wherein the support is selected from the group consisting of: beads, porous beads, magnetic beads, paramagnetic beads, porous matrices, arrays, surfaces, glass surfaces, silicon surfaces, plastic surfaces, slides, filters, nylon, chips, silicon crystal chips, flow chips, biochips comprising signal transduction electronic devices, wells, microtiter wells, plates, ELISA plates, disks, rotating interferometry disks, membranes, nitrocellulose membranes, nitrocellulose-based polymer surfaces, nanoparticles (e.g., comprising metals such as magnetic nanoparticles (Fe3O4), gold nanoparticles and / or silver nanoparticles), quantum dots, nanoshells, nanospheres, or any combination thereof.

[0341] Example 63'. The method of Example 62', wherein the support comprises sequencing beads, e.g., polystyrene beads, polymer beads, agarose beads, acrylamide beads, solid core beads, porous beads, magnetic beads, paramagnetic beads, glass beads, or controlled pore beads, or any combination thereof.

[0342] Example 64'. A method according to any of Examples 56'-63', wherein the average distance that each segment and its associated recording label are separated from other segments and their associated recording labels on the support is equal to or greater than about 20 nm, equal to or greater than about 50 nm, equal to or greater than about 100 nm, equal to or greater than about 150 nm, equal to or greater than about 200 nm, equal to or greater than about 250 nm, equal to or greater than about 300 nm, equal to or greater than about 350 nm, equal to or greater than about 400 nm, equal to or greater than about 450 nm, equal to or greater than about 500 nm, equal to or greater than about 550 nm, equal to or greater than about 600 nm, equal to or greater than about 650 nm, equal to or greater than about 700 nm, equal to or greater than about 750 nm, equal to or greater than about 800 nm, equal to or greater than about 850 nm, equal to or greater than about 900 nm, equal to or greater than about 950 nm, or equal to or greater than about 1 μm.

[0343] Example 65'. A method for analyzing multiple polypeptides, comprising: (a) immobilizing the multiple polypeptides to multiple substrates, wherein each substrate comprises a plurality of record tags, each record tag comprising a compartment tag, optionally wherein each compartment is a bead, a microfluidic droplet, a microwell, or a spaced area on a surface, or any combination thereof; (b) fragmenting the polypeptides immobilized on each substrate (e.g., by protease digestion) to produce a set of polypeptide fragments immobilized on the substrate; (c) contacting the immobilized set of polypeptide fragments with a library of binding agents, wherein each binding agent comprises a binding group and a coding tag, the coding tag comprising identification information about the binding group, wherein the binding group is capable of binding to one or more N-terminal, internal or C-terminal amino acids of the fragment, or is capable of binding to one or more N-terminal, internal, or C-terminal amino acids modified by a functionalizing agent; (d) allowing the transfer of information between (i) the record tag and (ii) the coding tag when the binding group binds to one or more N-terminal, internal or C-terminal amino acids of each fragment to produce extended record tags and / or extended coding tags; and (e) analyzing the extended record tags and / or extended coding tags.

[0344] Example 66'. The method of Example 65', wherein the multiple polypeptides having the same compartment tag belong to the same protein.

[0345] Example 67'. The method of Example 65', wherein the multiple polypeptides having the same compartment tag belong to different proteins, for example, two, three, four, five, six, seven, eight, nine, ten or more proteins.

[0346] Example 68'. The method of any of Examples 65' to 67', wherein each substrate defines a compartment.

[0347] Example 69'. A method according to any one of Examples 65' to 68', wherein the plurality of substrates are selected from the group consisting of beads, porous beads, porous matrices, arrays, surfaces, glass surfaces, silicon surfaces, plastic surfaces, slides, filters, nylon, chips, silicon crystal chips, flow chips, biochips comprising signal transduction electronic devices, wells, microtiter wells, plates, ELISA plates, disks, rotating interferometry disks, membranes, nitrocellulose membranes, nitrocellulose-based polymer surfaces, nanoparticles (e.g., comprising metals such as magnetic nanoparticles (Fe3O4), gold nanoparticles and / or silver nanoparticles), quantum dots, nanoshells, nanospheres, or any combination thereof.

[0348] Example 70'. A method according to any one of Examples 65'-69', wherein each of the multiple substrates comprises barcoded particles, such as barcoded beads, for example, polystyrene beads, polymer beads, agarose beads, acrylamide beads, solid core beads, porous beads, magnetic beads, paramagnetic beads, glass beads or controlled pore beads, or any combination thereof.

[0349] Example 71'. A method according to any one of Examples 50'-70', wherein the functionalizing agent comprises a chemical agent, an enzyme and / or a biological agent, such as 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.

[0350] Example 72'. A method according to any one of Examples 50'-71', wherein the record label comprises a nucleic acid, an oligonucleotide, a modified oligonucleotide, a DNA molecule, a DNA with pseudo-complementary bases, an RNA molecule, a BNA molecule, an XNA molecule, an LNA molecule, a PNA molecule, a γPNA molecule or a morpholino, or a combination thereof.

[0351] Example 73'. A method according to any one of Examples 50' to 72', wherein the record tag comprises a universal priming site; a priming site for amplification, sequencing, or both; optionally, a unique molecular identifier (UMI); a barcode; optionally, a spacer at its 3' end; or a combination thereof.

[0352] Example 74'. The method according to any one of Examples 50' to 73', which is used to determine the sequence of a polypeptide or multiple polypeptides.

[0353] Example 75'. A method according to any one of Examples 50'-74', wherein the encoding tag comprises a nucleic acid, an oligonucleotide, a modified oligonucleotide, a DNA molecule, a DNA with pseudo-complementary bases, an RNA molecule, a BNA molecule, an XNA molecule, an LNA molecule, a PNA molecule, a γPNA molecule or a morpholino, or a combination thereof.

[0354] Example 76'. The method of any one of Examples 50 to 75, wherein the encoding tag comprises an encoder sequence, an optional spacer, an optional unique molecular identifier (UMI), a universal priming site, or any combination thereof.

[0355] Example 77'. The method according to any one of Examples 50'-76', wherein the binding group and the coding tag are connected through a linker or a binding pair.

[0356] Example 78'. A method according to any one of Examples 50-77, wherein the binding group and the coding tag are connected by SpyTag-KTag / SpyLigase (wherein the two parts to be connected have a SpyTag / KTag pair, and SpyLigase connects the SpyTag to KTag, thereby connecting the two parts), a SnoopTag / SnoopCatcher peptide-protein pair, a sorting enzyme, or a HaloTag / HaloTag ligand pair, or any combination thereof.

[0357] Example 79'. A method according to any one of 1' to 78', wherein the encoding label and / or the recording label comprises one or more error correction codes, one or more encoder sequences, one or more barcodes, one or more UMIs, one or more compartment labels, or any combination thereof.

[0358] Example 80'. The method of Example 79', wherein the error correction code is selected from Hamming code, Lie distance code, asymmetric Lie distance code, Reed-Solomon code and Levenshtein-Tenengolts code.

[0359] Example 81'. The method of any one of Examples 1'-80', wherein analyzing the extended recording tag and / or the extended encoding tag comprises nucleic acid sequence analysis.

[0360] Example 82'. A method according to Example 81', wherein the nucleic acid sequence analysis comprises a nucleic acid sequencing method, such as sequencing by synthesis, sequencing by ligation, sequencing by hybridization, polony sequencing, ion semiconductor sequencing or pyrophosphate sequencing, or any combination thereof.

[0361] Example 83'. The method of Example 82', wherein the nucleic acid sequencing method is single-molecule real-time sequencing, nanopore-based sequencing, or direct imaging of DNA using advanced microscopy.

[0362] Example 84'. The method according to any one of Examples 1'-83' further comprises one or more washing steps.

[0363] Example 85'. The method of any of Examples 1'-84', wherein the extended recording tags and / or extended encoding tags are amplified prior to analysis.

[0364] Example 86'. The method of any one of Examples 1'-85', wherein the extended recording tag and / or extended encoding tag is subjected to a target enrichment assay prior to analysis.

[0365] Example 87'. The method of any one of Examples 1'-86', wherein the extended recording tag and / or extended coding tag is subjected to a subtraction assay prior to analysis.

[0366] In one aspect, disclosed herein are kits comprising: (a) a library of reagents, wherein each reagent comprises (i) a small molecule, a peptide or peptide mimetic, a peptide (e.g., a peptoid, a β-peptide, or a D-peptide mimetic), a polysaccharide, and / or an aptamer, and (ii) a coding tag comprising identification information about the small molecule, the peptide or peptide mimetic, the peptide (e.g., a peptoid, a β-peptide, or a D-peptide mimetic), a polysaccharide, or an aptamer; and optionally (b) a set of proteins, wherein each protein is directly or indirectly associated with a record tag, wherein each A library of proteins and / or their associated record tags or each agent is directly or indirectly immobilized on a support, and wherein the set of proteins, record tags, and agent libraries are configured to allow the transfer of information between (i) the record tags associated with each protein that binds and / or reacts with one or more small molecules, peptides or peptide mimetics, peptides (e.g., peptoids, β-peptides, or D-peptide peptide mimetics), polysaccharides, or aptamers of the agent and (ii) the coding tags of the one or more agents to generate extended record tags and / or extended coding tags.

[0367] In one aspect, disclosed herein is a kit for analyzing polypeptides, comprising: (a) a library of binding agents, wherein each binding agent comprises a binding group and a coding tag, the coding tag comprising identification information about the binding group, wherein the binding group is capable of binding to one or more N-terminal, internal or C-terminal amino acids of a fragment, or is capable of binding to one or more N-terminal, internal, or C-terminal amino acids modified by a functionalizing agent; and optionally (b) a set of fragments of the polypeptide, wherein each fragment is directly or indirectly associated with a recording tag, or (b') a tool for fragmenting the polypeptide, such as a protease, wherein each fragment and / or its associated recording tag, or each binding agent is directly or indirectly immobilized on a support, and wherein the set of fragments of the polypeptide, the recording tag and the library of binding agents are configured to allow the transfer of information between (i) the recording tag associated with each fragment and (ii) the coding tag upon binding between the binding group and one or more N-terminal, internal or C-terminal amino acids of the fragment to produce an extended recording tag and / or an extended coding tag.

[0368] In one aspect, disclosed herein is a kit for analyzing a plurality of polypeptides, comprising: (a) a library of binding agents, wherein each binding agent comprises a binding group and a coding tag, the coding tag comprising identification information about the binding group, wherein the binding group is capable of binding to one or more N-terminal, internal or C-terminal amino acids of a fragment, or is capable of binding to one or more N-terminal, internal, or C-terminal amino acids modified by a functionalizing agent; and (b) a plurality of substrates, optionally having a plurality of polypeptides immobilized thereon, wherein each substrate comprises a plurality of recording tags, each recording tag comprising a compartment tag, optionally wherein each compartment is a bead, a microfluidic droplet, a microwell, or a spaced area on a surface, or any combination thereof, wherein the polypeptides immobilized on each substrate are configured to be fragmented (e.g., by protease cleavage) to produce a set of polypeptide fragments immobilized on the substrate, wherein the plurality of polypeptides, the recording tags and the library of binding agents are configured to allow the transfer of information between (i) the recording tags and (ii) the coding tags upon binding between the binding group and one or more N-terminal, internal or C-terminal amino acids of each fragment to generate extended recording tags and / or extended coding tags.

[0369] The following aspects provide supplementary explanations of the present disclosure.

[0370] Aspect 1. A method for analyzing a polypeptide, comprising the following steps: (a) providing a polypeptide optionally directly or indirectly bound to a record tag; (b) functionalizing the N-terminal amino acid (NTAA) of the polypeptide with a chemical reagent, wherein the chemical reagent comprises a compound selected from the group consisting of: (i) a compound of formula (I)

[0371]

[0372] or a salt or conjugate thereof,

[0373] in,

[0374] R 1 and R 2 Each independently is H, C 1-6 Alkyl, cycloalkyl, -C(O)R a ,-C(O)OR b or -S(O)2R c ;

[0375] R a , R b and R c Each independently is H, C 1-6 Alkyl, C 1-6 haloalkyl, arylalkyl, aryl or heteroaryl, wherein C 1-6 Alkyl, C 1-6 haloalkyl, arylalkyl, aryl and heteroaryl are each unsubstituted or substituted;

[0376] R 3 is heteroaryl, -NR d C(O)OR e or –SR f , wherein heteroaryl is unsubstituted or substituted;

[0377] R d , R e and R f Each independently is H or C 1-6 alkyl; and

[0378] Optionally, when R 3 yes When R 1 and R 2 Not all are H;

[0379] (ii) Compounds of formula (II):

[0380]

[0381] or a salt or conjugate thereof,

[0382] in,

[0383] R 4 It is H, C 1-6 Alkyl, cycloalkyl, –C(O)R g OR –C(O)OR g ;and

[0384] R g H, C 1-6 Alkyl, C 2-6 Alkenyl, C1-6 Halogenated alkyl or arylalkyl, wherein C 1-6 Alkyl, C 2-6 Alkenyl, C 1-6 Haloalkyl and arylalkyl are each unsubstituted or substituted;

[0385] (iii) Compounds of formula (III):

[0386] R 5 -N=C=S (iii)

[0387] or a salt or conjugate thereof,

[0388] in,

[0389] R 5 It is C 1-6 Alkyl, C 2-6 alkenyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl;

[0390] Among them C 1-6 Alkyl, C 2-6 Alkenyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl are each unsubstituted or selected from halogen, -NR h R i , -S(O)2R j or one or more groups consisting of a heterocyclic group;

[0391] R h , R i and R j Each independently is H, C 1-6 Alkyl, C 1-6 haloalkyl, arylalkyl, aryl or heteroaryl, wherein C 1-6 Alkyl, C 1-6 Haloalkyl, arylalkyl, aryl and heteroaryl are each unsubstituted or substituted.

[0392] (iv) Compounds of formula (IV):

[0393]

[0394] or a salt or conjugate thereof,

[0395] in,

[0396] R 6 and R 7 Each independently is H, C 1-6 Alkyl, -CO2C 1-4 Alkyl, -OR k , aryl or cycloalkyl, wherein C 1-6 Alkyl, -CO2C 1-4 Alkyl, -ORk , aryl and cycloalkyl are each unsubstituted or substituted; and

[0397] R k H, C 1-6 Alkyl or heterocyclic group, wherein C 1-6 The alkyl group and the heterocyclyl group are each unsubstituted or substituted;

[0398] (v) Compounds of formula (V):

[0399]

[0400] or a salt or conjugate thereof,

[0401] in,

[0402] R 8 Is a halogen or –OR m ;

[0403] R m H, C 1-6 an alkyl or heterocyclic group; and

[0404] R 9 is hydrogen, halogen or C 1-6 alkyl halide;

[0405] (vi) Metal complex of formula (VI):

[0406] ML n (VI)

[0407] or a salt or conjugate thereof,

[0408] in,

[0409] M is a metal selected from the group consisting of Co, Cu, Pd, Pt, Zn and Ni;

[0410] L is a ligand selected from the group consisting of: -OH, -OH2, 2,2'-bipyridine (bpy), 1,5-dithiocyclooctane (dtco), 1,2-bis(diphenylphosphino)ethane (dppe), ethylenediamine (en) and triethylenetetramine (trien); and

[0411] n is an integer from 1 to 8, inclusive;

[0412] wherein each L may be the same or different; and

[0413] (vii) Compounds of formula (VII):

[0414]

[0415] or a salt or conjugate thereof,

[0416] in,

[0417] G 1 It is N, NR 13 or CR 13 R 14 ;

[0418] G 2 is N or CH;

[0419] p is 0 or 1;

[0420] R 10 , R 11 , R 12 , R 13 and R 14 Each independently selected from H, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 Alkylamine and C 1-6 Alkylhydroxylamine, where C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 Alkylamine and C 1-6 The alkylhydroxylamines are each unsubstituted or substituted, and R 10 and R 11 may optionally form a ring together; and

[0421] R 15 is H or OH,

[0422] (c) contacting the polypeptide with a first binding agent comprising a first binding moiety capable of binding to the functionalized NTAA;

[0423] (c1) a first coded label having identification information about the first binding agent, or

[0424] (c2) a first detectable label;

[0425] (d)(d1) transferring the information of the first coding tag to a recording tag to generate an extended recording tag and analyzing the extended recording tag, or

[0426] (d2) detecting the first detectable label;

[0427] Wherein step (b) is performed before step (c), after step (c) and before step (d), or after step (d).

[0428] In some examples, sequencing methods employ an "Edman-like" N-terminal amino acid degradation method. Edman-like degradation consists of two key steps: 1) functionalization of the -amine on the peptide's NTAA, and 2) elimination of the functionalized NTAA. Standard Edelman functionalization chemistry, as well as the Edelman-like functionalization chemistry described herein, exhibits poor functionalization and elimination of N-terminal proline residues. Consequently, the presence of N-terminal proline can cause a "pause" in the cycle sequencing reaction. Therefore, in some embodiments of the methods described herein, it is beneficial to remove any N-terminal proline at the beginning of each Edman-like degradation cycle by exposing the target polypeptide to a proline aminopeptidase that specifically cleaves only the N-terminal proline (proline iminopeptidase). Therefore, in some embodiments, each of the methods and assays described herein may optionally include an additional step of contacting the polypeptide being analyzed with a proline aminopeptidase. Similarly, kits for practicing these methods may optionally include at least one proline aminopeptidase.

[0429] Several proline aminopeptidases (PAPs) are known in the literature for this purpose. In a preferred embodiment, small monomeric PAPs (approximately 25-35 kDa) are used to remove NTAA prolines. Suitable monomeric PAPs for use in the methods and kits described herein include family members from Bacillus coagulans, L. delbrueckii, Neisseria gonorrhoeae, Flavobacterium meningosepticum, Serratia marcescens, and Lactobacillus plantarum (MEROPS S33.001) (Nakajima, Ito et al., 2006) (Kitazono, Yoshimoto et al., 1992). Suitable polymeric PAPs are also known and include an enzyme from D. hansenii (Bolumar, Sanz et al., 2003). Natural or engineered PAPs can be used. Effective mapping of peptide sequences without proline residues generated by the methods and assays herein can be accomplished by back-mapping the peptide reads to a "no proline" proteome. At the bioinformatics level, this essentially translates to proteins consisting of 19 amino acid residues instead of 20.

[0430] Alternatively, to preserve proline information, two binding steps can be employed before and after proline removal to enable detection of proline residues, but this is achieved at the additional cost of an additional binding / encoding cycle per sequencing cycle. Additionally, the concept of combining Edman-type chemistry with an R-group-specific aminopeptidase can be used to remove any NTF / NTE-resistant amino acid; however, in preferred embodiments, only a single ineffective amino residue, typically proline, is removed by the aminopeptidase. Removal of multiple residues results in a combinatorial explosion of removed sequences (i.e., removal of P and W results in the removal of sequences with runs of P, runs of W, and runs of P and W).

[0431] Aspect 2. The method according to aspect 1, wherein step (a) comprises providing the polypeptide and the associated record tag bound to a support (eg, a solid support).

[0432] Aspect 3. The method according to aspect 1, wherein step (a) comprises providing the polypeptide linked to the associated record tag in solution.

[0433] Aspect 4. The method according to aspect 1, wherein step (a) comprises providing a polypeptide indirectly associated with the record label.

[0434] Aspect 5. The method according to aspect 1, wherein in step (a) the polypeptide is not associated with a record tag.

[0435] Aspect 6. The method according to any one of aspects 1 to 5, wherein step (b) is performed before step (c).

[0436] Aspect 7. The method according to any one of aspects 1 to 5, wherein step (b) is performed after step (c) and before step (d).

[0437] Aspect 8. The method according to any one of aspects 1 to 5, wherein step (b) is performed after step (c) and step (d).

[0438] Aspect 9. The method according to any one of aspects 1-5, wherein steps (a), (b), (c1), and (d1) occur sequentially.

[0439] Aspect 10. The method according to any one of aspects 1-5, wherein steps (a), (b), (c), and (d1) occur sequentially.

[0440] Aspect 11. The method according to any one of aspects 1-5, wherein steps (a), (c1), (d1), and (b) occur sequentially.

[0441] Aspect 12. The method according to any one of aspects 1-5, wherein steps (a), (b), (c2), and (d2) occur in sequence.

[0442] Aspect 13. The method according to any one of aspects 1-5, wherein steps (a), (c2), (b), and (d2) occur in sequence.

[0443] Aspect 14. The method according to any one of aspects 1-5, wherein steps (a), (c2), (d2), and (b) occur in sequence.

[0444] Aspect 15. A method according to any one of Aspects 1-14, wherein step (c) further comprises contacting the polypeptide with a second (or higher-order) binding agent, the second (or higher-order) binding agent comprising a second (or higher-order) binding portion capable of binding to a functionalized NTAA different from the functionalized NTAA of step (b), and a coding tag having identification information about the second (or higher-order) binding agent.

[0445] Aspect 16. A method according to aspect 15, wherein after the polypeptide is contacted with the first binding agent, the polypeptide is then contacted with a second (or higher order) binding agent.

[0446] Aspect 17. The method according to aspect 15, wherein contacting the polypeptide with the second (or higher order) binding agent occurs simultaneously with contacting the polypeptide with the first binding agent.

[0447] Aspect 18. A method for analyzing a polypeptide, comprising the following steps:

[0448] (a) providing a polypeptide optionally directly or indirectly associated with a record tag;

[0449] (b) functionalizing the N-terminal amino acid (NTAA) of the polypeptide with a chemical reagent to produce a functionalized NTAA, wherein the chemical reagent comprises a compound selected from the group consisting of:

[0450] (i) Compound of formula (I):

[0451]

[0452] or a salt or conjugate thereof,

[0453] in,

[0454] R 1 and R 2 Each independently is H, C 1-6 Alkyl, cycloalkyl, -C(O)R a ,-C(O)OR b or -S(O)2R c ;

[0455] R a , R b and R c Each independently is H, C 1-6 Alkyl, C 1-6 haloalkyl, arylalkyl, aryl or heteroaryl, wherein C 1-6 Alkyl, C 1-6 haloalkyl, arylalkyl, aryl and heteroaryl are each unsubstituted or substituted;

[0456] R 3 is heteroaryl, -NRd C(O)OR e or –SR f , wherein heteroaryl is unsubstituted or substituted;

[0457] R d , R e and R f Each independently is H or C 1-6 alkyl; and

[0458] Optionally, when R 3 yes When R 1 and R 2 Not all are H;

[0459] (ii) Compounds of formula (II):

[0460]

[0461] or a salt or conjugate thereof,

[0462] in,

[0463] R 4 It is H, C 1-6 Alkyl, cycloalkyl, –C(O)R g OR –C(O)OR g ;and

[0464] R g H, C 1-6 Alkyl, C 2-6 Alkenyl, C 1-6 Halogenated alkyl or arylalkyl, wherein C 1-6 Alkyl, C 2-6 Alkenyl, C 1-6 Haloalkyl and arylalkyl are each unsubstituted or substituted;

[0465] (iii) Compounds of formula (III):

[0466] R5-N=C=S (III)

[0467] or a salt or conjugate thereof,

[0468] in,

[0469] R 5 It is C 1-6 Alkyl, C 2-6 alkenyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl;

[0470] Among them C 1-6 Alkyl, C 2-6Alkenyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl are each unsubstituted or selected from halogen, -NR h R i , -S(O)2R j or one or more groups consisting of a heterocyclic group;

[0471] R h , R i and R j Each independently is H, C 1-6 Alkyl, C 1-6 haloalkyl, arylalkyl, aryl or heteroaryl, wherein C 1-6 Alkyl, C 1-6 Haloalkyl, arylalkyl, aryl and heteroaryl are each unsubstituted or substituted.

[0472] (iv) Compounds of formula (IV):

[0473]

[0474] or a salt or conjugate thereof,

[0475] in,

[0476] R 6 and R 7 Each independently is H, C 1-6 Alkyl, -CO2C 1-4 Alkyl, -OR k , aryl or cycloalkyl, wherein C 1-6 Alkyl, -CO2C 1-4 Alkyl, -OR k , aryl and cycloalkyl are each unsubstituted or substituted; and

[0477] R k H, C 1-6 Alkyl or heterocyclic group, wherein C 1-6 Alkyl and heterocyclyl are each unsubstituted or substituted; and

[0478] (v) Compounds of formula (V):

[0479]

[0480] or a salt or conjugate thereof,

[0481] in,

[0482] R 8 Is a halogen or –OR m ;

[0483] R m H, C 1-6 an alkyl or heterocyclic group; and

[0484] R 9 is hydrogen, halogen or C 1-6 haloalkyl; and

[0485] (vi) Metal complex of formula (VI):

[0486] ML n (VI)

[0487] or a salt or conjugate thereof,

[0488] in,

[0489] M is a metal selected from the group consisting of Co, Cu, Pd, Pt, Zn and Ni;

[0490] L is a ligand selected from the group consisting of: -OH, -OH2, 2,2'-bipyridine (bpy), 1,5-dithiocyclooctane (dtco), 1,2-bis(diphenylphosphino)ethane (dppe), ethylenediamine (en) and triethylenetetramine (trien); and

[0491] n is an integer from 1 to 8, inclusive;

[0492] wherein each L may be the same or different; and

[0493] (vii) Compounds of formula (VII):

[0494]

[0495] or a salt or conjugate thereof,

[0496] in,

[0497] G 1 It is N, NR 13 or CR 13 R 14 ;

[0498] G 2 is N or CH;

[0499] p is 0 or 1;

[0500] R 10 , R 11 , R 12 , R 13 and R 14 Each independently selected from H, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 Alkylamine and C 1-6 Alkylhydroxylamine, where C 1-6 Alkyl, C1-6 Halogenated alkyl, C 1-6 Alkylamine and C 1-6 The alkylhydroxylamines are each unsubstituted or substituted, and R 10 and R 11 may optionally form a ring together; and

[0501] R 15 is H or OH,

[0502] (c) contacting the polypeptide with a first binding agent comprising a first binding moiety capable of binding to the functionalized NTAA and (c1) a first coding tag having identification information about the first binding agent, or (c2) a first detectable tag;

[0503] (d)(d1) transferring the information of the first coded label to a recording label to generate a first extended recording label and analyzing the extended recording label, or

[0504] (d2) detecting the first detectable label, and

[0505] (e) eliminating the functionalized NTAA to expose a new NTAA;

[0506] Wherein step (b) is performed before step (c), after step (c) and before step (d), or after step (d).

[0507] In some embodiments of compounds of Formula (I) for use in any of the methods and kits disclosed herein, R 3 In some embodiments of formula (I), R 3 is a 5- or 6-membered monocyclic heteroaryl. In some embodiments of formula (I), R 3 is a 5- or 6-membered monocyclic heteroaryl group containing one or more N. Preferably, R 3 is selected from pyrazole, imidazole, triazole and tetrazole, and is linked to the amidine of formula (I) via the nitrogen atom of the pyrazole, imidazole, triazole or tetrazole ring, and R 3 is optionally selected from halogen, C 1-3 Alkyl, C 1-3 In some embodiments, R 3 wherein G1 is N, CH or CX, wherein X is halogen, C 1-3 Alkyl, C 1-3 In some embodiments, R 3 for Or, wherein X is Me, F, Cl, CF3 or NO2. In some embodiments, R 3 for wherein G1 is N or CH. In some embodiments, R 3 yes In some embodiments, R 3 In some embodiments, R3 is a 9- or 10-membered bicyclic heteroaryl. 3 yes

[0508] Aspect 19. The method according to aspect 18, wherein step (a) comprises providing the polypeptide and the associated record tag bound to a support (eg, a solid support).

[0509] Aspect 20. The method according to aspect 18, wherein step (a) comprises providing the polypeptide linked to the associated record tag in solution.

[0510] Aspect 21. The method according to aspect 18, wherein step (a) comprises providing a polypeptide indirectly associated with the record label.

[0511] Aspect 22. The method according to aspect 18, wherein in step (a) the polypeptide is not associated with a record tag.

[0512] Aspect 23. The method according to any one of aspects 18-22, wherein step (b) is performed before step (c).

[0513] Aspect 24. The method according to any one of aspects 18-22, wherein step (b) is performed after step (c) and before step (d).

[0514] Aspect 25. The method according to any one of aspects 18-22, wherein step (b) is performed after step (c) and step (d).

[0515] Aspect 26. The method according to any one of aspects 18-22, wherein steps (a), (b), (c1) and (c2) occur in sequence.

[0516] Aspect 27. The method according to any one of aspects 18-22, wherein steps (a), (c1), (b), (d1) and (d1) occur in sequence.

[0517] Aspect 28. The method according to any one of aspects 18-22, wherein steps (a), (c1), (d1) and (b) occur sequentially.

[0518] Aspect 29. The method according to any one of aspects 18-22, wherein steps (a), (b), (c2) and (d2) occur in sequence.

[0519] Aspect 30. The method according to any one of aspects 18-22, wherein steps (a), (c2), (b) and (d2) occur sequentially.

[0520] Aspect 31. The method according to any one of aspects 18-22, wherein steps (a), (c2), (d2) and (b) occur in sequence.

[0521] Aspect 32. The method according to any one of aspects 18 to 31, further comprising the following steps:

[0522] (f) functionalizing the new NTAA of the polypeptide with a chemical reagent to produce a new functionalized NTAA;

[0523] (g) contacting the polypeptide with a second (or higher order) binding agent, the second (or higher order) binding agent comprising a second (or higher order) binding group capable of binding to the newly functionalized NTAA and (g1) a second coding tag having identification information about the second (or higher order) binding agent, or (g2) a second detectable tag;

[0524] (h)(h1) transferring the information of the second encoded tag to the first extended recording tag to generate a second extended recording tag, and analyzing the second extended recording tag, or

[0525] (h2) detecting a second detectable label, and

[0526] (i) removing the modified NTAA to expose a new NTAA;

[0527] Wherein step (f) is performed before step (g), after step (g) and before step (h), or after step (h).

[0528] In any of aspects 1-32, the method optionally further comprises the step of contacting the polypeptide with a proline aminopeptidase under conditions suitable to cleave any N-terminal proline present, typically prior to said method steps.

[0529] Aspect 33. The method of aspect 32, wherein the chemical agent comprises a compound selected from the group consisting of:

[0530] (i) Compound of formula (I):

[0531]

[0532] or a salt or conjugate thereof,

[0533] in,

[0534] R 1 and R 2 Each independently is H, C 1-6 Alkyl, cycloalkyl, -C(O)R a ,-C(O)OR b or -S(O)2Rc ;

[0535] R a , R b and R c Each independently is H, C 1-6 Alkyl, C 1-6 haloalkyl, arylalkyl, aryl or heteroaryl, wherein C 1-6 Alkyl, C 1-6 haloalkyl, arylalkyl, aryl and heteroaryl are each unsubstituted or substituted;

[0536] R 3 is heteroaryl, -NR d C(O)OR e or –SR f , wherein heteroaryl is unsubstituted or substituted;

[0537] R d , R e and R f Each independently is H or C 1-6 alkyl; and

[0538] Optionally, when R 3 yes When R 1 and R 2 Not all are H;

[0539] (ii) Compounds of formula (II):

[0540]

[0541] or a salt or conjugate thereof,

[0542] in,

[0543] R 4 It is H, C 1-6 Alkyl, cycloalkyl, –C(O)R g OR –C(O)OR g ;and

[0544] R g H, C 1-6 Alkyl, C 2-6 Alkenyl, C 1-6 Halogenated alkyl or arylalkyl, wherein C 1-6 Alkyl, C 2-6 Alkenyl, C 1-6 Haloalkyl and arylalkyl are each unsubstituted or substituted;

[0545] (iii) Compounds of formula (III):

[0546] R 5-N=C=S (III)

[0547] or a salt or conjugate thereof,

[0548] in,

[0549] R 5 It is C 1-6 Alkyl, C 2-6 alkenyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl;

[0550] Among them C 1-6 Alkyl, C 2-6 Alkenyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl are each unsubstituted or selected from halogen, -NR h R i , -S(O)2R j or one or more groups consisting of a heterocyclic group;

[0551] R h , R i and R j Each independently is H, C 1-6 Alkyl, C 1-6 haloalkyl, arylalkyl, aryl or heteroaryl, wherein C 1-6 Alkyl, C 1-6 Haloalkyl, arylalkyl, aryl and heteroaryl are each unsubstituted or substituted.

[0552] (iv) Compounds of formula (IV):

[0553]

[0554] or a salt or conjugate thereof,

[0555] in,

[0556] R 6 and R 7 Each independently is H, C 1-6 Alkyl, -CO2C 1-4 Alkyl, -OR k , aryl or cycloalkyl, wherein C 1-6 Alkyl, -CO2C 1-4 Alkyl, -OR k , aryl and cycloalkyl are each unsubstituted or substituted; and

[0557] R k H, C 1-6 Alkyl or heterocyclic group, wherein C 1-6 Alkyl and heterocyclyl are each unsubstituted or substituted; and

[0558] (v) Compounds of formula (V):

[0559]

[0560] or a salt or conjugate thereof,

[0561] in,

[0562] R 8 Is a halogen or –OR m ;

[0563] R m H, C 1-6 an alkyl or heterocyclic group; and

[0564] R 9 is hydrogen, halogen or C 1-6 haloalkyl; and

[0565] (vi) Metal complex of formula (VI):

[0566] ML n (VI)

[0567] or a salt or conjugate thereof,

[0568] in,

[0569] M is a metal selected from the group consisting of Co, Cu, Pd, Pt, Zn and Ni;

[0570] L is a ligand selected from the group consisting of: -OH, -OH2, 2,2'-bipyridine (bpy), 1,5-dithiocyclooctane (dtco), 1,2-bis(diphenylphosphino)ethane (dppe), ethylenediamine (en) and triethylenetetramine (trien); and

[0571] n is an integer from 1 to 8, inclusive;

[0572] wherein each L may be the same or different; and

[0573] (vii) Compounds of formula (VII):

[0574]

[0575] or a salt or conjugate thereof,

[0576] in,

[0577] G 1 It is N, NR 13 or CR 13 R 14 ;

[0578] G 2 is N or CH;

[0579] p is 0 or 1;

[0580] R 10 , R 11 , R 12 , R 13 and R 14 Each independently selected from H, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 Alkylamine and C 1-6 Alkylhydroxylamine, where C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 Alkylamine and C 1-6 The alkylhydroxylamines are each unsubstituted or substituted, and R 10 and R 11 may optionally form a ring together; and

[0581] R 15 is H or OH.

[0582] Aspect 34. The method according to aspect 32 or aspect 33, wherein step (f) is performed before step (g).

[0583] Aspect 35. The method according to aspect 32 or aspect 33, wherein step (f) is performed after step (g) and before step (h).

[0584] Aspect 36. The method according to aspect 32 or aspect 33, wherein step (f) is performed after step (g) and step (h).

[0585] Aspect 37. The method of aspect 32 or aspect 33, wherein steps (f), (g1) and (h1) occur sequentially.

[0586] Aspect 38. The method of aspect 32 or aspect 33, wherein steps (g1), (f) and (h1) occur sequentially.

[0587] Aspect 39. The method of aspect 32 or aspect 33, wherein steps (g1), (h1) and (f) occur sequentially.

[0588] Aspect 40. The method of aspect 32 or aspect 33, wherein steps (f), (g2) and (h2) occur sequentially.

[0589] Aspect 41. The method of aspect 32 or aspect 33, wherein steps (g2), (f) and (h2) occur sequentially.

[0590] Aspect 42. The method of aspect 32 or aspect 33, wherein steps (g2), (h2) and (f) occur sequentially.

[0591] Aspect 43. The method according to aspects 32-42, wherein after contacting the peptide with the first binding agent, the peptide is subsequently contacted with a second (or higher order) binding agent.

[0592] Aspect 44. The method according to aspects 32-42, wherein contacting the polypeptide with the second (or higher order) binding agent occurs simultaneously with contacting the polypeptide with the first binding agent.

[0593] Aspect 45. The method according to any one of claims 1 to 44, wherein the polypeptide is obtained by fragmenting a protein from a biological sample.

[0594] Aspect 46. A method according to any one of Aspects 1-45, wherein the record tag comprises a nucleic acid, an oligonucleotide, a modified oligonucleotide, a DNA molecule, a DNA with pseudo-complementary bases, a DNA with protected bases, an RNA molecule, a BNA molecule, an XNA molecule, an LNA molecule, a PNA molecule, a γPNA molecule or a morpholino DNA, or a combination thereof.

[0595] Aspect 47. The method according to aspect 46, wherein the DNA molecule is backbone-modified, sugar-modified or nucleobase-modified.

[0596] Aspect 48. A method according to aspect 46, wherein the DNA molecule has a nucleobase protecting group, such as Alloc, an electrophilic protecting group, such as thiranes, an acetyl protecting group, a nitrobenzyl protecting group, a sulfonate protecting group, or a traditional base-labile protecting group, including Ultramild reagent.

[0597] Aspect 49. The method according to any one of aspects 1 to 48, wherein the record label comprises a universal priming site.

[0598] Aspect 50. The method according to aspect 49, wherein the universal priming site comprises a primer site for amplification, sequencing, or both.

[0599] Aspect 51. The method according to any one of aspects 1-50, wherein the record tag comprises a unique molecular identifier (UMI).

[0600] Aspect 52. The method according to any one of aspects 1-51, wherein the record label comprises a bar code.

[0601] Aspect 53. The method according to any one of aspects 1 to 52, wherein the record tag comprises a spacer at its 3'-end.

[0602] Aspect 54. The method according to any one of aspects 1 to 53, wherein the polypeptide and the associated record tag are covalently bound to a solid support.

[0603] Aspect 55. According to any of the methods described in Aspects 1-54, the 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.

[0604] Aspect 56. The method according to aspect 55, wherein the support comprises gold, silver, a semiconductor, or quantum dots.

[0605] Aspect 57. The method of aspect 55, wherein the nanoparticles comprise gold, silver, or quantum dots.

[0606] Aspect 58. The method according to aspect 55, wherein the 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.

[0607] Aspect 59. The method according to any one of aspects 1 to 58, wherein the polypeptide and the associated record label are attached to a solid support.

[0608] Aspect 60. The method of aspect 59, wherein the plurality of polypeptides are spaced apart on the support, wherein the average distance between the polypeptides is about ≥ 20 nm.

[0609] Aspect 61. The method according to any one of aspects 1-60, wherein the binding portion of the binding agent comprises a peptide or protein.

[0610] Aspect 62. The method according to any one of aspects 1-61, wherein the binding portion of the binding agent comprises an aminopeptidase or a variant, mutant or modified protein thereof; an aminoacyl-tRNA synthetase or a variant, mutant or modified protein thereof; an anticalin or a variant, mutant or modified protein thereof; a Clps (e.g., ClpS2) or a variant, mutant or modified protein thereof; a UBR box protein or a variant, mutant or modified protein thereof; or a modified small molecule that binds to an amino acid, i.e., vancomycin or a variant, mutant or modified molecule thereof; or an antibody or a binding fragment thereof; or any combination thereof.

[0611] Aspect 63. The method of any one of Aspects 1-62, wherein the binding agent binds to a single amino acid residue (e.g., an N-terminal amino acid residue, a C-terminal amino acid residue, or an internal amino acid residue), a dipeptide (e.g., an N-terminal dipeptide, a C-terminal dipeptide, or an internal dipeptide), a tripeptide (e.g., an N-terminal tripeptide, a C-terminal tripeptide, or an internal tripeptide), or a post-translationally modified polypeptide.

[0612] Aspect 64. The method according to any one of aspects 1-62, wherein the binding agent binds to an NTAA-functionalized single amino acid residue, an NTAA-functionalized dipeptide, an NTAA-functionalized tripeptide, or an NTAA-functionalized polypeptide.

[0613] Aspect 65. The method according to any one of aspects 1 to 64, wherein the binding portion of the binding agent is capable of selectively binding to the polypeptide.

[0614] Aspect 66. The method according to any one of aspects 1 to 65, wherein the encoding 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.

[0615] Aspect 67. The method according to any one of aspects 1-66, wherein the encoded tag comprises an encoder or barcode sequence.

[0616] Aspect 68. The method according to any one of aspects 1-67, wherein the encoding tag further comprises a spacer, a binding cycle specific sequence, a unique molecular identifier, a universal priming site, or any combination thereof.

[0617] Aspect 69. The method according to any one of aspects 1 to 68, wherein the binding moiety and the coding tag are connected by a linker.

[0618] Aspect 70. The method according to aspects 1-69, wherein the binding moiety and the encoding tag are linked via a SpyTag / SpyCatcher peptide-protein pair, a SnoopTag / SnoopCatcher peptide-protein pair or a HaloTag / HaloTag ligand pair.

[0619] Aspect 71. The method according to any one of aspects 1 to 70, wherein the information transfer from the recording tag to the encoding tag is mediated by DNA ligase or RNA ligase.

[0620] Aspect 72. The method according to any one of aspects 1 to 70, wherein the information transfer from the encoding tag to the recording tag is mediated by DNA polymerase, RNA polymerase or reverse transcriptase.

[0621] Aspect 73. The method according to any one of aspects 1 to 70, wherein the information transfer from the recording tag to the encoding tag is mediated by chemical ligation.

[0622] Aspect 74. The method according to aspect 73, wherein chemical ligation is performed using single-stranded DNA.

[0623] 75. The method of aspect 74, wherein chemical ligation is performed using double-stranded DNA.

[0624] 76. The method of any one of aspects 1-72, wherein analyzing the extended record tag comprises a nucleic acid sequencing method.

[0625] 77. The method of aspect 76, wherein the nucleic acid sequencing method is sequencing by synthesis, sequencing by ligation, sequencing by hybridization, polony sequencing, ion semiconductor sequencing or pyrosequencing.

[0626] 78. The method of aspect 76, wherein the nucleic acid sequencing method is single molecule real-time sequencing, nanopore-based sequencing, or direct imaging of DNA using advanced microscopy.

[0627] 79. The method according to any one of aspects 1 to 78, wherein the extended record tags are amplified prior to analysis.

[0628] 80. The method according to any one of aspects 1-79, further comprising the step of adding a cycle marker.

[0629] 81. A method according to aspect 80, wherein the cycle signature provides information about the order in which the binding agent binds to the polypeptide.

[0630] 82. A method according to aspect 80 or aspect 81, wherein a loop marker is added to the encoding tag.

[0631] 83. A method according to clause 80 or clause 81, wherein a loop marker is added to the record tag.

[0632] 84. The method according to aspect 80 or aspect 81, wherein a circulating label is added to the binding agent.

[0633] 85. A method according to aspect 80 or aspect 81, wherein the circulation mark is added independently of the encoding label, the recording label and the binding agent.

[0634] 86. The method according to any one of aspects 1 to 85, wherein the order of the encoded tag information contained in the extended record tags provides information about the binding order of the binding agents to the macromolecule.

[0635] 87. A method according to any one of aspects 1 to 86, wherein the frequency of the encoded tag information contained in the extended record tag provides information about the binding frequency of the binding agent to the polypeptide.

[0636] 88. A method according to any one of aspects 1 to 87, wherein a plurality of extended record tags representing a plurality of polypeptides are analysed in parallel.

[0637] 89. The method of aspect 88, wherein a plurality of extended record tags representing a plurality of polypeptides are analyzed in a multiplexed assay.

[0638] 90. The method of any one of aspects 88 or 89, wherein the plurality of extended record tags are subjected to target abundance detection prior to analysis.

[0639] 91. The method of any one of aspects 88-90, wherein the plurality of extended record tags are subjected to a subtraction assay prior to analysis.

[0640] 92. The method of any one of aspects 88-91, wherein the plurality of extended record tags are normalized prior to analysis to reduce high abundance species.

[0641] 93. The method according to any one of aspects 1-92, wherein NTAA is removed from the polypeptide by chemical cleavage or enzymatic cleavage.

[0642] 94. The method of aspect 93, wherein NTAA is eliminated by a carboxypeptidase or an aminopeptidase, or a variant, mutant, or modified protein thereof; a hydrolase, or a variant, mutant, or modified protein thereof; mild Edman degradation; an Edmanase enzyme; TFA, alkali; or any combination thereof.

[0643] 95. The method of aspect 94, wherein the mild Edman degradation uses dichloro or monochloro acid.

[0644] 96. The method of aspect 94, wherein the mild Edman degradation uses TFA, TCA, or DCA.

[0645] 97. The method of aspect 94, wherein the mild Edman degradation uses triethylammonium acetate.

[0646] 98. The method of aspect 94, wherein the base is a hydroxide, an alkylated amine, a cyclic amine, a carbonate buffer, or a metal salt.

[0647] 99. The method of aspect 98, wherein the hydroxide is sodium hydroxide.

[0648] 100. The method of aspect 98, wherein the alkylated amine is selected from the group consisting of methylamine, ethylamine, propylamine, dimethylamine, diethylamine, dipropylamine, trimethylamine, triethylamine, tripropylamine, cyclohexylamine, benzylamine, aniline, diphenylamine, N,N-diisopropylethylamine (DIPEA) and lithium diisopropylamide (LDA).

[0649] 101. The method of aspect 98, wherein the cyclic amine is selected from pyridine, pyrimidine, imidazole, pyrrole, indole, piperidine, pyrrolidine, 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) and 1,5-diazabicyclo[4.3.0]non-5-ene (DBN).

[0650] 102. The method of aspect 98, wherein the carbonate buffer comprises sodium carbonate, potassium carbonate, calcium carbonate, sodium bicarbonate, potassium bicarbonate, or calcium bicarbonate.

[0651] 103. The method of aspect 98, wherein the metal salt comprises silver.

[0652] 104. The method of aspect 103, wherein the metal salt is AgClO4.

[0653] 105. The method according to any one of aspects 1-104, wherein at least one binding agent binds to the terminal amino acid residue, the terminal two amino acid residues or the terminal three amino acid residues.

[0654] 106. The method according to any one of aspects 1-105, wherein at least one binding agent binds to a post-translationally modified amino acid.

[0655] 107. The method according to any one of aspects 1-106, wherein the chemical agent comprises a compound selected from the group consisting of compounds of formula (IV):

[0656]

[0657] or a salt or conjugate thereof,

[0658] in,

[0659] R 1 and R 2 Each independently is H, C 1-6 Alkyl, cycloalkyl, -C(O)R a ,-C(O)OR b or -S(O)2R c ;

[0660] R a , R b and R c Each independently is H, C 1-6 Alkyl, C1-6 haloalkyl, arylalkyl, aryl or heteroaryl, wherein C 1-6 Alkyl, C 1-6 haloalkyl, arylalkyl, aryl and heteroaryl are each unsubstituted or substituted;

[0661] R 3 is heteroaryl, -NR d C(O)OR e or –SR f , wherein heteroaryl is unsubstituted or substituted;

[0662] R d , R e and R f Each independently is H or C 1-6 alkyl; and

[0663] Optionally wherein when R3 is When R 1 and R 2 Not all are H.

[0664] 108. The method of aspect 107, wherein R 1 yes

[0665] 109. The method according to aspect 107 or 108, wherein R 2 yes

[0666] 110. The method according to any one of aspects 107-109, wherein R 1 or R 2 for

[0667] 111. The method according to any one of aspects 107-110, wherein R 3 yes wherein G1 is N or CH.

[0668] 112. The method according to any one of claims 107-110, wherein R 3 yes

[0669] 113. The method according to aspect 107, wherein the compound of formula (I) is selected from the group consisting of: as well as and optionally further comprising a compound selected from the group consisting of: (N-Boc, N'-trifluoroacetyl-pyrazolecarboxamidine, N,N'-diacetyl-pyrazolecarboxamidine, N-methyl-pyrazolecarboxamidine, N,N'-diacetyl-N-methyl-pyrazolecarboxamidine, N,N'-diacetyl-N-methyl-pyrazolecarboxamidine, N,N'-diacetyl-N-methyl-4-nitro-pyrazolecarboxamidine, and N,N'-diacetyl-N-methyl-4-trifluoromethyl-pyrazolecarboxamidine, or a salt or conjugate thereof.

[0670] 114. The method according to any one of aspects 107-113, wherein the chemical reagent further comprises Mukaiyama reagent (2-chloro-1-methylpyridinium iodide).

[0671] 115. The method according to any one of aspects 1-106, wherein the chemical agent comprises a compound selected from the group consisting of compounds of formula (II):

[0672]

[0673] or a salt or conjugate thereof,

[0674] in,

[0675] R 4 It is H, C 1-6 Alkyl, cycloalkyl, –C(O)R g OR –C(O)OR g ;and

[0676] R g H, C 1-6 Alkyl, C 2-6 Alkenyl, C 1-6 Halogenated alkyl or arylalkyl, wherein C 1-6 Alkyl, C 2-6 Alkenyl, C 1-6 Haloalkyl and arylalkyl are each unsubstituted or substituted;

[0677] 116. The method of aspect 115, wherein R 4 It is carboxybenzyl.

[0678] 117. The method of aspect 115, wherein R 4 Yes – C(O)R g , and R g It is C 2-6 alkenyl, which is optionally substituted with aryl, heteroaryl or heterocycloalkyl.

[0679] 118. The method according to aspect 115, wherein the compound is selected from the group consisting of:

[0680] as well as or a salt or conjugate thereof.

[0681] 119. The method according to any one of aspects 115-118, wherein the chemical reagent further comprises TMS-Cl, Sc(OTf)2, Zn(OTf)2 or a lanthanide-containing reagent.

[0682] 120. The method according to any one of aspects 1 to 106, wherein the chemical agent comprises a compound from the group consisting of compounds of formula (III):

[0683] R 5 -N=C=S (III)

[0684] or a salt or conjugate thereof,

[0685] in,

[0686] R 5 It is C 1-6 Alkyl, C 2-6 alkenyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl;

[0687] Among them C 1-6 Alkyl, C 2-6 Alkenyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl are each unsubstituted or selected from halogen, -NR h R i , -S(O)2R j or one or more groups consisting of a heterocyclic group;

[0688] R h , R i and R j Each independently is H, C 1-6 Alkyl, C 1-6 haloalkyl, arylalkyl, aryl or heteroaryl, wherein C 1-6 Alkyl, C 1-6 haloalkyl, arylalkyl, aryl and heteroaryl are each unsubstituted or substituted;

[0689] 121. The method of aspect 120, wherein R 5 is a substituted phenyl group.

[0690] 122. The method according to aspect 120 or aspect 121, wherein R 5 is phenyl, which is replaced by one or more selected from halogen, -NR h R i 、-S(O)2R j and heterocyclic groups.

[0691] 123. The method of aspect 120, wherein the compound of formula (III) is trimethylsilyl isothiocyanate (TMSITC) or pentafluorophenyl isothiocyanate (PFPITC).

[0692] 124. The method according to any one of aspects 120-123, wherein the chemical reagent further comprises a carbodiimide compound.

[0693] 125. The method according to any one of aspects 120 to 124, wherein NTAA is eliminated using trifluoroacetic acid or hydrochloric acid.

[0694] 126. The method according to any one of aspects 120-124, wherein NTAA is eliminated using mild Edman degradation.

[0695] 127. The method according to any one of aspects 120 to 124, wherein NTAA is eliminated using Edmanase or an engineered hydrolase, aminopeptidase or carboxypeptidase.

[0696] 128. The method according to any one of aspects 1-106, wherein the chemical agent comprises a compound selected from the group consisting of compounds of formula (IV):

[0697]

[0698] or a salt or conjugate thereof,

[0699] in,

[0700] R 6 and R 7 Each independently is H, C 1-6 Alkyl, -CO2C 1-4 Alkyl, -OR k , aryl or cycloalkyl, wherein C 1-6 Alkyl, -CO2C 1-4 Alkyl, -OR k , aryl and cycloalkyl are each unsubstituted or substituted; and

[0701] R k H, C 1-6 Alkyl or heterocyclic group, wherein C 1-6 The alkyl group and the heterocyclic group are each unsubstituted or substituted.

[0702] 129. The method of aspect 128, wherein R 6 and R 7 Each independently is H, C 1-6 Alkyl or cycloalkyl.

[0703] 130. The method of aspect 128, wherein the compound is selected from the group consisting of: as well as or a salt or conjugate thereof.

[0704] 131. The method according to any one of aspects 128-130, wherein the compound of formula (IV) is prepared by desulfurization of the corresponding thiourea.

[0705] 132. The method according to any one of aspects 128-131, wherein the chemical reagent further comprises Mukaiyama reagent (2-chloro-1-methylpyridinium iodide).

[0706] 133. The method according to any one of aspects 128-131, wherein the chemical reagent further comprises a Lewis acid.

[0707] 134. The method of aspect 133, wherein the Lewis acid is selected from N-((aryl)imino-acetonaphthone)ZnCl2, Zn(OTf)2, ZnCl2, PdCl2, CuCl and CuCl2.

[0708] 135. The method according to any one of aspects 1-106, wherein the chemical agent comprises a compound selected from the group consisting of compounds of formula (V):

[0709]

[0710] or a salt or conjugate thereof,

[0711] in,

[0712] R 8 Is a halogen or –OR m ;

[0713] R m H, C 1-6 an alkyl or heterocyclic group; and

[0714] R 9 is hydrogen, halogen or C 1-6 Halogenated alkyl.

[0715] 136. The method of aspect 135, wherein R 8 It's chlorine.

[0716] 137. The method according to aspect 135 or aspect 136, wherein R 9 It is hydrogen or bromine.

[0717] 138. The method according to any one of aspects 135 to 137, wherein the chemical reagent further comprises a peptide coupling reagent.

[0718] 139. The method of aspect 138, wherein the peptide coupling reagent is a carbodiimide compound.

[0719] 140. The method of aspect 139, wherein the carbodiimide compound is diisopropylcarbodiimide (DIC) or 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC).

[0720] 141. The method according to any one of aspects 135 to 140, wherein NTAA is eliminated using acyl peptide hydrolase (APH).

[0721] 142. The method according to any one of aspects 1-106, wherein the chemical agent comprises a metal complex of formula (VI):

[0722] ML n (VI)

[0723] or a salt or conjugate thereof,

[0724] in,

[0725] M is a metal selected from the group consisting of Co, Cu, Pd, Pt, Zn and Ni;

[0726] L is a ligand selected from the group consisting of: -OH, -OH2, 2,2'-bipyridine (bpy), 1,5-dithiocyclooctane (dtco), 1,2-bis(diphenylphosphino)ethane (dppe), ethylenediamine (en) and triethylenetetramine (trien); and

[0727] n is an integer from 1 to 8, inclusive;

[0728] Each L may be the same or different.

[0729] 143. The method of aspect 142, wherein M is Co.

[0730] 144. The method of aspect 142 or aspect 143, wherein the chemical reagent comprises cis-β-hydroxy(triethylenetetramine) hydrated cobalt(III) complex.

[0731] 145. The method of aspect 142, wherein the chemical agent comprises β-[Co(trien)(OH)(OH2)] 2+ .

[0732] 146. The method according to any one of aspects 1-106, wherein the chemical agent comprises a compound selected from the group consisting of compounds of formula (VII):

[0733]

[0734] or a salt or conjugate thereof,

[0735] in,

[0736] G 1 It is N, NR 13 or CR 13 R 14 ;

[0737] G 2 is N or CH;

[0738] p is 0 or 1;

[0739] R 10 , R 11 , R 12 , R 13 and R 14 Each independently selected from H, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 Alkylamine and C 1-6 Alkylhydroxylamine, where C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 Alkylamine and C 1-6 The alkylhydroxylamines are each unsubstituted or substituted, and R 10 and R 11 may optionally form a ring together; and

[0740] R 15 is H or OH.

[0741] 147. The method of aspect 146, wherein G 1 is CH2 and G 2 Is CH, or G 1 is NH and G 2 It's N.

[0742] 148. The method of aspect 146 or 147, wherein R 12 It’s H.

[0743] 149. The method according to any one of aspects 146-148, wherein R 10 and R 11 Each is H.

[0744] 150. The method according to aspect 146, wherein the compound of formula (VII) is selected from the group consisting of: as well as or a salt or conjugate thereof.

[0745] 151. The method according to any one of aspects 107-150, wherein NTAA is eliminated using a base.

[0746] 152. The method of aspect 151, wherein the base is a hydroxide, an alkylated amine, a cyclic amine, a carbonate buffer, or a metal salt.

[0747] 153. The method of aspect 152, wherein the hydroxide is sodium hydroxide.

[0748] 154. The method of aspect 152, wherein the alkylated amine is selected from methylamine, ethylamine, propylamine, dimethylamine, diethylamine, dipropylamine, trimethylamine, triethylamine, tripropylamine, cyclohexylamine, benzylamine, aniline, diphenylamine, N,N-diisopropylethylamine (DIPEA), and lithium diisopropylamide (LDA).

[0749] 155. The method of aspect 152, wherein the cyclic amine is selected from pyridine, pyrimidine, imidazole, pyrrole, indole, piperidine, pyrrolidine, 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) and 1,5-diazabicyclo[4.3.0]non-5-ene (DBN).

[0750] 156. The method of aspect 152, wherein the carbonate buffer comprises sodium carbonate, potassium carbonate, calcium carbonate, sodium bicarbonate, potassium bicarbonate, or calcium bicarbonate.

[0751] 157. The method of aspect 152, wherein the metal salt comprises silver.

[0752] 158. The method of aspect 152, wherein the metal salt is AgClO4.

[0753] 159. The method according to any one of aspects 1-158, wherein the chemical agent comprises a conjugate selected from the group consisting of:

[0754]

[0755] where R 1 , R 2 and R 3 As defined for formula (I) in aspect 1, and Q is a ligand;

[0756]

[0757] where R 4 As defined for formula (II) in aspect 1, and Q is a ligand;

[0758]

[0759] where R 5 As defined for formula (III) in aspect 1, and Q is a ligand;

[0760]

[0761] where R 6 and R 7 As defined for formula (IV) in aspect 1, and Q is a ligand;

[0762]

[0763] where R 8 and R 9 As defined for formula (V) in aspect 1, and Q is a ligand;

[0764] (ML n )-Q formula (VI)-Q,

[0765] wherein M, L and N are as defined for formula (V) in aspect 1, and Q is a ligand;

[0766]

[0767] where R 10 , R 11 , R 12 , R 15 , G 1 , G 2 and p are as defined in Aspect 1 for Formula (VII), and Q is a ligand.

[0768] 160. The method according to aspect 159, wherein Q is selected from -C 1-6 Alkyl, -C 2-6 Alkenyl, -C 2-6 Alkynyl, aryl, heteroaryl, heterocyclyl, -N=C=S, -CN, -C(O)R n ,-C(O)OR o , --SR p or -S(O)2R q ; where -C 1-6 Alkyl, -C 2-6 Alkenyl, -C 2-6 Alkynyl, aryl, heteroaryl and heterocyclyl are each unsubstituted or substituted, and R n , R o , R p and R q Each independently selected from -C 1-6 Alkyl, -C 1-6 Haloalkyl, -C 2-6 Alkenyl, -C 2-6Alkynyl, aryl, heteroaryl and heterocyclyl.

[0769] 161. The method of aspect 159, wherein Q is selected from the group consisting of: as well as

[0770] 162. The method of aspect 159, wherein Q is a fluorophore.

[0771] 163. The method according to any one of aspects 1-162, wherein step (b) of aspect 1 or aspect 5 comprises difunctionalization of NTAA.

[0772] 164. The method of aspect 163, wherein step (b) of aspect 1 or aspect 5 comprises difunctionalizing NTAA with (1) a first chemical reagent and (2) a second chemical reagent, the first chemical reagent comprising a compound selected from the group consisting of: a compound of formula (I), (II), (III), (IV), (V), (VI) and (VII), or a salt or conjugate thereof.

[0773] 165. The method of aspect 164, wherein the second chemical agent comprises a compound of formula (VIIIa) or (VIIIb):

[0774]

[0775] or a salt or conjugate thereof,

[0776] in,

[0777] R 13 It is H, C 1-6 Alkyl, aryl, heteroaryl, cycloalkyl or heterocycloalkyl, wherein C 1-6 Alkyl, aryl, heteroaryl, cycloalkyl and heterocycloalkyl are each unsubstituted or substituted; or

[0778] R 13 -X (VIIIb)

[0779] in,

[0780] R 13 C 1-6 alkyl, aryl, heteroaryl, cycloalkyl or heterocycloalkyl, each of which is unsubstituted or substituted; and

[0781] X is a halogen.

[0782] 166. The method of aspect 164 or 165, wherein step (b) of aspect 1 or aspect 5 comprises functionalizing the NTAA with a second chemical reagent prior to functionalizing with the first chemical reagent.

[0783] 167. The method of aspect 164 or 165, wherein step (b) of aspect 1 or aspect 5 comprises functionalizing the NTAA with a first chemical reagent prior to functionalizing with a second chemical reagent.

[0784] 168. The method of any one of aspects 32-167, wherein step (f) of aspect 6 comprises difunctionalization of NTAA.

[0785] 169. The method of aspect 168, wherein step (f) of aspect 6 comprises difunctionalizing NTAA with (1) a first chemical reagent and (2) a second chemical reagent, the first chemical reagent comprising a compound selected from the group consisting of: compounds of formula (I), (II), (III), (IV), (V), (VI), (VII) and (VIII).

[0786] 170. The method of aspect 169, wherein the second chemical agent comprises a compound of formula (VIIIa) or (VIIIb):

[0787]

[0788] or a salt or conjugate thereof,

[0789] in,

[0790] R 13 It is H, C 1-6 Alkyl, aryl, heteroaryl, cycloalkyl or heterocycloalkyl, wherein C 1-6 Alkyl, aryl, heteroaryl, cycloalkyl and heterocycloalkyl are each unsubstituted or substituted; or

[0791] R 13 -X (VIIIb)

[0792] in,

[0793] R 13 C 1-6 alkyl, aryl, heteroaryl, cycloalkyl or heterocycloalkyl, each of which is unsubstituted or substituted; and

[0794] X is a halogen.

[0795] 171. The method of aspect 169 or 170, wherein step (b) of aspect 1 or aspect 5 comprises functionalizing the NTAA with a second chemical reagent prior to functionalizing with the first chemical reagent.

[0796] 172. The method of aspect 169 or 170, wherein step (b) of aspect 1 or aspect 5 comprises functionalizing the NTAA with a first chemical reagent prior to functionalizing with a second chemical reagent.

[0797] 173. The method according to any one of aspects 165-167 and 170-172, wherein the compound of formula (VIIIa) is formaldehyde.

[0798] 174. The method according to any one of aspects 165-167 and 170-172, wherein the compound of formula (VIIIb) is iodomethane.

[0799] 175. A method of analyzing one or more polypeptides from a sample comprising a plurality of protein complexes, proteins, or polypeptides, the method comprising:

[0800] (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 support (e.g., a solid support), wherein the plurality of compartment tags within a single compartment are identical and different from the compartment tags of other compartments;

[0801] (b) fragmenting the plurality of protein complexes, proteins and / or polypeptides into a plurality of polypeptides;

[0802] (c) contacting the plurality of polypeptides with the plurality of compartment tags under conditions sufficient to allow annealing or ligation of the plurality of polypeptides to the plurality of compartment tags within the plurality of compartments, thereby producing a plurality of compartment-tagged polypeptides;

[0803] (d) collecting compartment-tagged polypeptides from the plurality of compartments; and

[0804] (e) Analyzing one or more compartment-tagged polypeptides according to the method of any one of aspects-174.

[0805] 176. The method of aspect 175, wherein the compartment is a microfluidic droplet.

[0806] 177. The method of aspect 175, wherein the compartment is a microwell.

[0807] 178. The method of aspect 175, wherein the compartments are separate areas on a plane.

[0808] 179. The method of any one of aspects 175-178, wherein each compartment contains on average a single cell.

[0809] 180. A method of analyzing one or more polypeptides from a sample comprising a plurality of protein complexes, proteins, or polypeptides, the method comprising:

[0810] (a) labeling multiple protein complexes, proteins or polypeptides with multiple universal DNA tags;

[0811] (b) partitioning the 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 identical and different from the compartment tags of other compartments;

[0812] (c) contacting the plurality of protein complexes, proteins, or polypeptides with the plurality of compartment tags under conditions sufficient to allow annealing or ligation of the plurality of peptides to the plurality of compartment tags within the plurality of compartments, thereby producing a plurality of compartment-tagged protein complexes, proteins, or polypeptides;

[0813] (d) collecting compartment-tagged protein complexes, proteins, or polypeptides from the plurality of compartments;

[0814] (e) optionally fragmenting the compartment-tagged protein complex, protein or polypeptide into compartment-tagged polypeptides; and

[0815] (f) analysing one or more compartment-tagged polypeptides according to the method of any one of aspects 1-174.

[0816] 181. A method according to any one of aspects 175 to 180, wherein the compartment label information is transferred to a record tag associated with the polypeptide by primer extension or ligation.

[0817] 182. According to any one of aspects 1-181, 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.

[0818] 183. The method according to any one of aspects 175 to 181, wherein the solid support is a bead.

[0819] 184. The method of aspect 183, 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.

[0820] 185. The method of any one of aspects 175 to 184, wherein the compartment tag comprises a single-stranded or double-stranded nucleic acid molecule.

[0821] 186. The method of any one of aspects 175-185, wherein the compartment label comprises a barcode and optionally a UMI.

[0822] 187. The method of aspect 186, wherein the solid support is a bead and the compartment label comprises a barcode, further wherein the bead comprising the plurality of compartment labels attached thereto is formed by split and pool synthesis.

[0823] 188. The method of aspect 186, wherein the solid support is a bead and the compartment labels comprise a barcode, further wherein the bead comprising the plurality of compartment labels attached thereto is formed by a single synthesis or immobilization.

[0824] 189. The method of any one of aspects 175-188, wherein the compartment tag is a component of a record tag, the record tag optionally further comprising a spacer, a barcode sequence, a unique molecular identifier, a universal priming site, or any combination thereof.

[0825] 190. The method of any one of claims 175-189, wherein the compartment tag further comprises a functional portion capable of reacting with an internal amino acid, a peptide backbone, or an N-terminal amino acid of a plurality of protein complexes, proteins, or polypeptides.

[0826] 191. The method of aspect 190, wherein the functional moiety is an aldehyde, an azide / alkyne, or a maleimide / thiol, or an epoxide / nucleophile, or an inverse electron demand Diels-Alder (iEDDA) group.

[0827] 192. The method of aspect 190, wherein the functional moiety is an aldehyde group.

[0828] 193. The method of any one of aspects 175-192, wherein the plurality of compartment labels are formed by printing, spotting, inkjet printing the compartment labels into the compartments, or a combination thereof.

[0829] 194. The method of any one of aspects 175-193, wherein the compartment tag further comprises a polypeptide.

[0830] 195. The method of aspect 194, wherein the compartment tag polypeptide further comprises a protein ligase recognition sequence.

[0831] 196. The method of aspect 195, wherein the protein ligase is butelase I or a homologue thereof.

[0832] 197. The method according to any one of aspects 175 to 196, wherein the plurality of polypeptides are fragmented using a protease.

[0833] 198. The method of aspect 197, wherein the protease is a metalloprotease.

[0834] 199. The method of aspect 198, wherein the activity of the metalloprotease is modulated by photon-stimulated release of metal cations.

[0835] 200. The method according to any one of aspects 175 to 199, further comprising subtracting one or more abundant proteins from the sample before partitioning the plurality of polypeptides into the plurality of compartments.

[0836] 201. The method according to any one of aspects 175 to 200, further comprising releasing the compartment tag from the solid support before attaching the plurality of polypeptides to the compartment tag.

[0837] 202. The method of aspect 175, further comprising the step of (d) linking the compartment-labeled polypeptide to a solid support associated with the record label.

[0838] 203. The method of aspect 202, further comprising transferring information of the compartment label on the compartment-tagged polypeptide to an associated record label.

[0839] 204. The method of aspect 203, further comprising removing the compartment tag from the compartment-tagged polypeptide prior to step (e).

[0840] 205. The method according to any one of aspects 175 to 204, further comprising determining the identity of the single cell from which the analyzed polypeptide originated based on the compartment tag sequence of the analyzed polypeptide.

[0841] 206. The method according to any one of aspects 175 to 204, further comprising determining the identity of the source protein or protein complex of the analyzed polypeptide based on the compartment tag sequence of the analyzed polypeptide.

[0842] 207. The method according to any one of aspects 32 to 206, wherein steps (f), (g), (h), and (i) are repeated with a plurality of amino acids.

[0843] 208. The method of aspect 207, wherein steps (f), (g), (h), and (i) are repeated with two or more amino acids.

[0844] 209. The method of aspect 207, wherein steps (f), (g), and (h) are repeated with up to about 100 amino acids.

[0845] 210. A kit for analyzing a polypeptide, comprising:

[0846] (a) a reagent for providing a polypeptide optionally directly or indirectly associated with a record tag;

[0847] (b) a reagent for functionalizing the N-terminal amino acid (NTAA) of a polypeptide, wherein the reagent comprises a compound selected from the group consisting of:

[0848] (i) Compound of formula (I):

[0849]

[0850] or a salt or conjugate thereof,

[0851] in,

[0852] R 1 and R 2 Each independently is H, C 1-6 Alkyl, cycloalkyl, -C(O)R a ,-C(O)OR b or -S(O)2R c ;

[0853] R a , R b and R c Each independently is H, C 1-6 Alkyl, C 1-6 haloalkyl, arylalkyl, aryl or heteroaryl, wherein C 1-6 Alkyl, C 1-6 haloalkyl, arylalkyl, aryl and heteroaryl are each unsubstituted or substituted;

[0854] R 3 is heteroaryl, -NR d C(O)OR e or –SR f , wherein heteroaryl is unsubstituted or substituted;

[0855] R d , R e and R f Each independently is H or C 1-6 alkyl; and

[0856] Optionally, when R 3 yes When R 1 and R 2 Not all are H;

[0857] (ii) Compounds of formula (II):

[0858]

[0859] or a salt or conjugate thereof,

[0860] in,

[0861] R4 It is H, C 1-6 Alkyl, cycloalkyl, –C(O)R g OR –C(O)OR g ;and

[0862] R g H, C 1-6 Alkyl, C 2-6 Alkenyl, C 1-6 Halogenated alkyl or arylalkyl, wherein C 1-6 Alkyl, C 2-6 Alkenyl, C 1-6 Haloalkyl and arylalkyl are each unsubstituted or substituted;

[0863] (iii) Compounds of formula (III):

[0864] R 5 -N=C=S (III)

[0865] or a salt or conjugate thereof,

[0866] in,

[0867] R 5 It is C 1-6 Alkyl, C 2-6 alkenyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl;

[0868] Among them C 1-6 Alkyl, C 2-6 Alkenyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl are each unsubstituted or selected from halogen, -NR h R i , -S(O)2R j or one or more groups consisting of a heterocyclic group;

[0869] R h , R i and R j Each independently is H, C 1-6 Alkyl, C 1-6 haloalkyl, arylalkyl, aryl or heteroaryl, wherein C 1-6 Alkyl, C 1-6 Haloalkyl, arylalkyl, aryl and heteroaryl are each unsubstituted or substituted.

[0870] (iv) Compounds of formula (IV):

[0871]

[0872] or a salt or conjugate thereof,

[0873] in,

[0874] R 6 and R 7 Each independently is H, C 1-6 Alkyl, -CO2C 1-4 Alkyl, -OR k , aryl or cycloalkyl, wherein C 1-6 Alkyl, -CO2C 1-4 Alkyl, -OR k , aryl and cycloalkyl are each unsubstituted or substituted; and

[0875] R k H, C 1-6 Alkyl or heterocyclic group, wherein C 1-6 The alkyl group and the heterocyclyl group are each unsubstituted or substituted;

[0876] (v) Compounds of formula (V):

[0877]

[0878] or a salt or conjugate thereof,

[0879] in,

[0880] R 8 Is a halogen or –OR m ;

[0881] R m H, C 1-6 an alkyl or heterocyclic group; and

[0882] R 9 is hydrogen, halogen or C 1-6 alkyl halide;

[0883] (vi) Metal complex of formula (VI):

[0884] ML n (VI)

[0885] or a salt or conjugate thereof,

[0886] in,

[0887] M is a metal selected from the group consisting of Co, Cu, Pd, Pt, Zn and Ni;

[0888] L is a ligand selected from the group consisting of: -OH, -OH2, 2,2'-bipyridine (bpy), 1,5-dithiocyclooctane (dtco), 1,2-bis(diphenylphosphino)ethane (dppe), ethylenediamine (en) and triethylenetetramine (trien); and

[0889] n is an integer from 1 to 8, inclusive;

[0890] wherein each L may be the same or different; and

[0891] (vii) Compounds of formula (VII):

[0892]

[0893] or a salt or conjugate thereof,

[0894] in,

[0895] G 1 It is N, NR 13 or CR 13 R 14 ;

[0896] G 2 is N or CH;

[0897] p is 0 or 1;

[0898] R 10 , R 11 , R 12 , R 13 and R 14 Each independently selected from H, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 Alkylamine and C 1-6 Alkylhydroxylamine, where C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 Alkylamine and C 1-6 The alkylhydroxylamines are each unsubstituted or substituted, and R 10 and R 11 may optionally form a ring together; and

[0899] R 15 is H or OH;

[0900] (c) a first binding agent comprising a first binding moiety capable of binding to the functionalized NTAA and (c1) a first coded tag having identification information about the first binding agent, or (c2) a first detectable tag; and

[0901] (d) a reagent that transfers information of the first encoding tag to the recording tag to produce an extended recording tag; and

[0902] (e) A reagent for analyzing the extended recording label or a reagent for detecting the first detectable label.

[0903] 211. The kit of aspect 210, wherein the reagents of (a) are configured to provide the polypeptide and associated record tag attached to a support (eg, a solid support).

[0904] 212. The kit of aspect 210, wherein the reagent of (a) is configured to provide the polypeptide directly associated with the record tag in solution.

[0905] 213. The kit of aspect 210, wherein the reagent of (a) is configured to provide a polypeptide indirectly associated with the record tag.

[0906] 214. The kit of aspect 210, wherein the reagent of (a) is configured to provide a polypeptide that is not associated with a record tag.

[0907] 215. The kit according to any one of aspects 210-214, wherein the kit comprises two or more different reagents for functionalizing the NTAA of a polypeptide.

[0908] 216. The kit of aspect 215, wherein the kit comprises a first reagent and a second reagent, the first reagent comprising a compound selected from the group consisting of: a compound of formula (I), (II), (III), (IV), (V), (VI) and (VII) as described in aspect 125, or a salt or conjugate thereof.

[0909] 217. The kit of aspect 216, wherein the second reagent comprises a compound of formula (VIIIa) or (VIIIb):

[0910]

[0911] or a salt or conjugate thereof,

[0912] in,

[0913] R 13 It is H, C 1-6 Alkyl, aryl, heteroaryl, cycloalkyl or heterocycloalkyl, wherein C 1-6 Alkyl, aryl, heteroaryl, cycloalkyl and heterocycloalkyl are each unsubstituted or substituted; or

[0914] R 13 -XX (VIIIb)

[0915] in,

[0916] R 13 C 1-6 alkyl, aryl, heteroaryl, cycloalkyl or heterocycloalkyl, each of which is unsubstituted or substituted; and

[0917] X is a halogen.

[0918] 218. The kit according to aspect 217, wherein the compound of formula (VIIIa) is formaldehyde.

[0919] 219. The kit according to aspect 217, wherein the compound of formula (VIIIb) is iodomethane.

[0920] 220. The kit according to any one of aspects 210 to 219, wherein the kit comprises two or more different binding agents.

[0921] 221. The kit according to any one of aspects 210-220, further comprising a reagent for removing the functionalized NTAA to expose a new NTAA.

[0922] 222. The kit according to any one of aspects 211-221, wherein the kit comprises two or more different reagents for eliminating functionalized NTAA.

[0923] 223. The kit of aspect 221 or aspect 222, wherein the reagent for eliminating functionalized NTAA comprises a chemical cleavage reagent or an enzymatic cleavage reagent.

[0924] 224. A kit according to aspect 221 or aspect 222, wherein the reagent for eliminating functionalized NTAA comprises a carboxypeptidase or an aminopeptidase or a variant, mutant or modified protein thereof; a hydrolase or a variant, mutant or modified protein thereof; a mild Edman degradation reagent; an Edmanase enzyme; TFA; a base; or any combination thereof.

[0925] 225. The kit according to any one of aspects 210 to 224, wherein the chemical reagent comprises a compound selected from the group consisting of compounds of formula (I):

[0926]

[0927] or a salt or conjugate thereof,

[0928] in,

[0929] R 1 and R 2 Each independently is H, C 1-6 Alkyl, cycloalkyl, -C(O)R a ,-C(O)OR b or -S(O)2R c ;

[0930] R a , R b and R c Each independently is H, C 1-6 Alkyl, C 1-6 haloalkyl, arylalkyl, aryl or heteroaryl, wherein C 1-6 Alkyl, C1-6 haloalkyl, arylalkyl, aryl and heteroaryl are each unsubstituted or substituted;

[0931] R 3 is heteroaryl, -NR d C(O)OR e or –SR f , wherein heteroaryl is unsubstituted or substituted;

[0932] R d , R e and R f Each independently is H or C 1-6 alkyl; and

[0933] Optionally, when R 3 yes When R 1 and R 2 Not all are H;

[0934] 226. The kit according to aspect 225, wherein R 1 yes

[0935] 227. The kit according to aspect 225 or aspect 226, wherein R2 is

[0936] 228. The kit according to any one of aspects 225-227, wherein R 1 or R 2 yes

[0937] 229. The kit according to any one of aspects 225-228, wherein R 3 yes wherein G1 is N or CH.

[0938] 230. The kit according to any one of aspects 225-228, wherein R 3 yes

[0939] 231. The kit according to aspect 225, wherein the compound of formula (I) is selected from as well as or a salt or conjugate thereof.

[0940] 232. The kit according to any one of aspects 225 to 231, wherein the chemical reagents further comprise Mukaiyama reagent (2-chloro-1-methylpyridinium iodide).

[0941] 233. The kit according to any one of aspects 210 to 224, wherein the chemical reagent comprises a compound selected from the group consisting of compounds of formula (II):

[0942]

[0943] or a salt or conjugate thereof,

[0944] in,

[0945] R 4 It is H, C 1-6 Alkyl, cycloalkyl, –C(O)R g OR –C(O)OR g ;and

[0946] R g H, C 1-6 Alkyl, C 2-6 Alkenyl, C 1-6 Halogenated alkyl or arylalkyl, wherein C 1-6 Alkyl, C 2-6 Alkenyl, C 1-6 Haloalkyl and arylalkyl are each unsubstituted or substituted;

[0947] 234. The kit according to aspect 142, wherein R 4 It is carboxybenzyl.

[0948] 235. The kit according to aspect 142, wherein R 4 Yes – C(O)R g , and R g It is C 2-6 alkenyl, which is optionally substituted with aryl, heteroaryl or heterocycloalkyl.

[0949] 236. The kit according to aspect 142, wherein the compound is selected from as well as or a salt or conjugate thereof.

[0950] 237. The kit of any one of aspects 142-145, wherein the chemical reagent further comprises TMS-Cl, Sc(OTf)2, Zn(OTf)2 or a lanthanide-containing reagent.

[0951] 238. The kit according to any one of aspects 210 to 224, wherein the chemical reagent comprises a compound selected from the group consisting of compounds of formula (III):

[0952] R 5 -N=C=S (III)

[0953] or a salt or conjugate thereof,

[0954] in,

[0955] R 5 It is C 1-6 Alkyl, C 2-6 alkenyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl;

[0956] Among them C 1-6 Alkyl, C 2-6 Alkenyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl are each unsubstituted or selected from halogen, -NR h R i , -S(O)2Rj or one or more groups consisting of a heterocyclic group;

[0957] R h , R i and Rj are each independently H, C 1-6 Alkyl, C 1-6 haloalkyl, arylalkyl, aryl or heteroaryl, wherein C 1-6 Alkyl, C 1-6 haloalkyl, arylalkyl, aryl and heteroaryl are each unsubstituted or substituted;

[0958] 239. The kit according to aspect 238, wherein R 5 is a substituted phenyl group.

[0959] 240. The kit according to aspect 238 or aspect 239, wherein R 5 is phenyl, which is selected from halogen, -NR h R i , -S(O)2Rj and one or more groups consisting of a heterocyclic group.

[0960] 241. The kit of aspect 238, wherein the compound of formula (III) is trimethylsilyl isothiocyanate (TMSITC) or pentafluorophenyl isothiocyanate (PFPITC).

[0961] 242. The kit according to any one of aspects 238-240, wherein the chemical reagent further comprises a carbodiimide compound.

[0962] 243. The kit according to any one of aspects 238 to 242, wherein the reagent for eliminating functionalized NTAA comprises trifluoroacetic acid or hydrochloric acid.

[0963] 244. The kit of any one of aspects 238-243, wherein the reagent for eliminating functionalized NTAA comprises a mild Edman degradation reagent.

[0964] 245. The kit of any one of aspects 238-243, wherein the reagent for removing functionalized NTAA comprises Edmanase or an engineered hydrolase, aminopeptidase or carboxypeptidase.

[0965] 246. The kit according to any one of aspects 210-224, wherein the chemical reagent comprises a compound selected from the group consisting of compounds of formula (IV):

[0966]

[0967] or a salt or conjugate thereof,

[0968] in,

[0969] R 6 and R 7 Each independently is H, C 1-6 Alkyl, -CO2C 1-4 Alkyl, -OR k , aryl or cycloalkyl, wherein C 1-6 Alkyl, -CO2C 1-4 Alkyl, -OR k , aryl and cycloalkyl are each unsubstituted or substituted; and

[0970] R k H, C 1-6 Alkyl or heterocyclic group, wherein C 1-6 The alkyl group and the heterocyclic group are each unsubstituted or substituted.

[0971] 247. The kit according to aspect 246, wherein R 6 and R 7 Each independently is H, C 1-6 Alkyl or cycloalkyl.

[0972] 248. The kit according to aspect 246, wherein the compound is selected from as well as or a salt or conjugate thereof.

[0973] 249. The method according to any one of aspects 246-248, wherein the compound of formula (IV) is prepared by desulfurization of the corresponding thiourea.

[0974] 250. The kit according to any one of aspects 246 to 249, wherein the chemical reagents further comprise Mukaiyama reagent (2-chloro-1-methylpyridinium iodide).

[0975] 251. The kit of any one of aspects 246-249, wherein the chemical reagent further comprises a Lewis acid.

[0976] 252. The kit of aspect 251, wherein the Lewis acid is selected from N-((aryl)imino-acetonaphthone)ZnCl2, Zn(OTf)2, ZnCl2, PdCl2, CuCl2 and CuCl2.

[0977] 253. The kit according to any one of aspects 210-224, wherein the chemical reagent comprises a compound selected from the group consisting of compounds of formula (V):

[0978]

[0979] or a salt or conjugate thereof,

[0980] in,

[0981] R 8 Is a halogen or –OR m ;

[0982] R m H, C 1-6 an alkyl or heterocyclic group; and

[0983] R 9 is hydrogen, halogen or C 1-6 Halogenated alkyl.

[0984] 254. The kit according to aspect 253, wherein R 8 It's chlorine.

[0985] 255. The kit according to aspect 253 or aspect 254, wherein R 9 It is hydrogen or bromine.

[0986] 256. The kit according to any one of aspects 253 to 255, wherein the chemical reagents further comprise a peptide coupling agent.

[0987] 257. The kit of aspect 256, wherein the peptide coupling reagent is a carbodiimide compound.

[0988] 258. The kit of aspect 257, wherein the carbodiimide compound is diisopropylcarbodiimide (DIC) or 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC).

[0989] 259. The kit of any one of aspects 253 to 258, wherein the reagent for eliminating functionalized NTAA comprises acyl peptide hydrolase (APH).

[0990] 260. The method according to any one of aspects 210 to 224, wherein the chemical agent comprises a metal complex of formula (VI):

[0991] ML n (VI)

[0992] or a salt or conjugate thereof,

[0993] in,

[0994] M is a metal selected from the group consisting of Co, Cu, Pd, Pt, Zn and Ni;

[0995] L is a ligand selected from the group consisting of: -OH, -OH2, 2,2'-bipyridine (bpy), 1,5-dithiocyclooctane (dtco), 1,2-bis(diphenylphosphino)ethane (dppe), ethylenediamine (en) and triethylenetetramine (trien); and

[0996] n is an integer from 1 to 8, inclusive;

[0997] Each L may be the same or different.

[0998] 261. The kit of aspect 260, wherein M is Co.

[0999] 262. The kit of aspect 260 or 261, wherein the chemical reagent comprises cis-β-hydroxy(triethylenetetramine) hydrated cobalt(III) complex.

[1000] 263. The kit of aspect 260 or 261, wherein the chemical reagent comprises β-[Co(trien)(OH)(OH2)] 2+ .

[1001] 264. The kit according to any one of aspects 210 to 224, wherein the chemical reagent comprises a compound selected from the group consisting of compounds of formula (VII):

[1002]

[1003] or a salt or conjugate thereof,

[1004] in,

[1005] G 1 It is N, NR 13 or CR 13 R 14 ;

[1006] G 2 is N or CH;

[1007] p is 0 or 1;

[1008] R 10 , R 11 , R 12 , R 13 and R 14 Each independently selected from H, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 Alkylamine and C 1-6 Alkylhydroxylamine, where C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 Alkylamine and C 1-6 The alkylhydroxylamines are each unsubstituted or substituted, and R 10 and R 11 may optionally form a ring together; and

[1009] R 15 is H or OH.

[1010] 265. The kit according to aspect 264, wherein G 1 is CH2 and G 2 Is CH, or G 1 is NH and G 2 It's N.

[1011] 266. The kit according to aspect 264 or 265, wherein R 12 It’s H.

[1012] 267. The kit according to any one of aspects 264 to 266, wherein R 10 and R 11 Each is H.

[1013] 268. The kit of aspect 264, wherein the compound of formula (VII) is selected from the group consisting of: as well as or a salt or conjugate thereof.

[1014] 269. The kit according to any one of aspects 225-268, wherein the reagent for eliminating functionalized NTAA comprises a base.

[1015] 270. The kit of aspect 269, wherein the base is a hydroxide, an alkylated amine, a cyclic amine, a carbonate buffer, or a metal salt.

[1016] 271. The kit according to aspect 270, wherein the hydroxide is sodium hydroxide

[1017] 272. The kit of aspect 270, wherein the alkylated amine is selected from methylamine, ethylamine, propylamine, dimethylamine, diethylamine, dipropylamine, trimethylamine, triethylamine, tripropylamine, cyclohexylamine, benzylamine, aniline, diphenylamine, N,N-diisopropylethylamine (DIPEA), and lithium diisopropylamide (LDA).

[1018] 273. The kit according to aspect 270, wherein the cyclic amine is selected from pyridine, pyrimidine, imidazole, pyrrole, indole, piperidine, pyrrolidine, 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) and 1,5-diazabicyclo[4.3.0]non-5-ene (DBN).

[1019] 274. The kit of aspect 270, wherein the carbonate buffer comprises sodium carbonate, potassium carbonate, calcium carbonate, sodium bicarbonate, potassium bicarbonate or calcium bicarbonate.

[1020] 275. The kit of aspect 270, wherein the metal salt comprises silver.

[1021] 276. The kit of aspect 270, wherein the metal salt is AgClO4.

[1022] 277. The kit according to any one of aspects 210 to 276, wherein the chemical reagent comprises a conjugate selected from the group consisting of:

[1023]

[1024] where R 1 , R 2 and R 3 As defined for formula (I) in aspect 1, and Q is a ligand;

[1025]

[1026] where R 4 As defined for formula (II) in aspect 1, and Q is a ligand;

[1027]

[1028] where R 5 As defined for formula (III) in aspect 1, and Q is a ligand;

[1029]

[1030] where R 6 and R 7 As defined for formula (IV) in aspect 1, and Q is a ligand;

[1031]

[1032] where R 8 and R 9 As defined for formula (V) in aspect 1, and Q is a ligand;

[1033] (ML n )-Q formula (VI)-Q,

[1034] wherein M, L and N are as defined for formula (V) in aspect 1, and Q is a ligand;

[1035]

[1036] where R 10 , R 11 , R 12 , R 15 , G 1 , G 2 and p are as defined in Aspect 1 for Formula (VII), and Q is a ligand.

[1037] 278. The kit according to aspect 277, wherein Q is selected from -C 1-6 Alkyl, -C 2-6 Alkenyl, -C 2-6 Alkynyl, aryl, heteroaryl, heterocyclyl, -N=C=S, -CN, -C(O)R n ,-C(O)OR o , -SR p or -S(O)2R q ; where -C 1-6 Alkyl, -C 2-6 Alkenyl, -C 2-6 Alkynyl, aryl, heteroaryl and heterocyclyl are each unsubstituted or substituted, and R n , R o , R p and R q Each independently selected from -C 1-6 Alkyl, -C 1-6 Haloalkyl, -C 2-6 Alkenyl, -C 2-6 Alkynyl, aryl, heteroaryl and heterocyclyl.

[1038] 279. The kit of aspect 277, wherein Q is selected from the group consisting of: as well as

[1039] 280. The kit of aspect 277, wherein Q is a fluorophore.

[1040] 281. The kit according to any one of aspects 210 to 280, wherein the binding agent binds to the terminal amino acid residue, the terminal two amino acid residues or the terminal three amino acid residues.

[1041] 282. The method according to any one of aspects 210 to 280, wherein the binding agent binds to a post-translationally modified amino acid.

[1042] 283. A kit according to any one of aspects 210-282, wherein the record tag comprises a nucleic acid, an oligonucleotide, a modified oligonucleotide, a DNA molecule, a DNA with pseudo-complementary bases, a DNA with protected bases, an RNA molecule, a BNA molecule, an XNA molecule, an LNA molecule, a PNA molecule, a γPNA molecule or a morpholino DNA, or a combination thereof.

[1043] 284. The kit of aspect 283, wherein the DNA molecule is backbone-modified, sugar-modified or nucleobase-modified.

[1044] 285. The kit of aspect 283, wherein the DNA molecule has a nucleobase protecting group such as Alloc, an electrophilic protecting group such as thiranes, an acetyl protecting group, a nitrobenzyl protecting group, a sulfonate protecting group, or a traditional base labile protecting group including Ultramild reagent.

[1045] 286. The kit according to any one of aspects 210 to 285, wherein the record tag comprises a universal priming site.

[1046] 287. The kit of aspect 286, wherein the universal priming site comprises primer sites for amplification, sequencing, or both.

[1047] 288. The kit of any one of aspects 210-287, wherein the record tag comprises a unique molecular identifier (UMI).

[1048] 289. The kit according to any one of aspects 210 to 288, wherein the record label comprises a bar code.

[1049] 290. The kit according to any one of aspects 210 to 288, wherein the record tag comprises a spacer at its 3'-end.

[1050] 291. A kit according to any one of aspects 210 to 290, wherein the reagents for providing the polypeptide and the associated record tag attached to the support provide for covalent attachment of the polypeptide to the associated record tag on the support.

[1051] 292. According to the kit of any one of aspects 210-291, 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.

[1052] 293. The kit of aspect 292, wherein the carrier comprises gold, silver, a semiconductor, or quantum dots.

[1053] 294. The kit of aspect 292, wherein the nanoparticles comprise gold, silver, or quantum dots.

[1054] 295. The kit of aspect 292, 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.

[1055] 296. A kit according to any one of aspects 210 to 295, wherein the reagents for providing a polypeptide and an associated record tag attached to a support provide a plurality of polypeptides and associated record tags attached to a support.

[1056] 297. The kit of aspect 296, wherein the plurality of polypeptides are spaced apart on the support, wherein the average distance between the polypeptides is about ≥ 20 nm.

[1057] 298. The kit of any one of aspects 210 to 297, wherein the binding agent is a polypeptide or protein.

[1058] 299. The kit of any one of aspects 210-298, wherein the binding agent comprises an aminopeptidase or a variant, mutant or modified protein thereof; an aminoacyl-tRNA synthetase or a variant, mutant or modified protein thereof; an avidin or a variant, mutant or modified protein thereof; a ClpS or a variant, mutant or modified protein thereof; or a modified small molecule that binds to an amino acid, i.e., vancomycin or a variant, mutant or modified molecule thereof; or an antibody or a binding fragment thereof; or any combination thereof.

[1059] 300. The kit of any of aspects 210-299, wherein the binding agent binds to a single amino acid residue (e.g., an N-terminal amino acid residue, a C-terminal amino acid residue, or an internal amino acid residue), a dipeptide (e.g., an N-terminal dipeptide, a C-terminal dipeptide, or an internal dipeptide), a tripeptide (e.g., an N-terminal tripeptide, a C-terminal tripeptide, or an internal tripeptide), or a post-translational modification of the analyte or polypeptide.

[1060] 301. The kit according to any one of aspects 210 to 300, wherein the binding agent binds to an NTAA-functionalized single amino acid residue, an NTAA-functionalized dipeptide, an NTAA-functionalized tripeptide, or an NTAA-functionalized polypeptide.

[1061] 302. The kit of any one of aspects 210 to 301, wherein the binding agent is capable of selectively binding to the polypeptide.

[1062] 303. The kit according to any one of aspects 210-302, wherein the encoding tag is a DNA molecule, an RNA molecule, a BNA molecule, an XNA molecule, an LNA molecule, a PNA molecule, a gamma PNA molecule or a combination thereof.

[1063] 304. The kit of any one of aspects 210-303, wherein the encoding tag comprises an encoder or barcode sequence.

[1064] 305. The kit according to any one of aspects 210 to 304, wherein the encoding tag further comprises a spacer, a binding cycle specific sequence, a unique molecular identifier, a universal priming site, or any combination thereof.

[1065] 306. The kit according to any one of aspects 210 to 305, wherein the binding moiety and the coding tag in the binding agent are connected by a linker.

[1066] 307. The kit according to any one of aspects 210 to 305, wherein the binding moiety and the encoding tag are linked via a SpyTag / SpyCatcher peptide-protein pair, a SnoopTag / SnoopCatcher peptide-protein pair or a HaloTag / HaloTag ligand pair.

[1067] 308. The kit according to any one of aspects 210 to 307, wherein the information transfer from the recording tag to the encoding tag is mediated by DNA ligase or RNA ligase.

[1068] 309. The kit according to any one of aspects 210 to 307, wherein the information transfer from the encoding tag to the recording tag is mediated by DNA polymerase, RNA polymerase or reverse transcriptase.

[1069] 310. The kit according to any one of aspects 210 to 307, wherein the reagents for transferring information from the encoding tag to the recording tag comprise chemical ligation reagents.

[1070] 311. The kit of aspect 310, wherein the chemical ligation reagent is for single-stranded DNA.

[1071] 312. The kit of aspect 310, wherein the chemical ligation reagent is for double-stranded DNA.

[1072] 313. The kit according to any one of aspects 210 to 312, further comprising a ligation reagent consisting of two DNA or RNA ligase variants, an adenylating variant and a constitutively non-adenylated variant.

[1073] 314. The kit according to any one of aspects 210 to 312, further comprising a ligation reagent consisting of a DNA or RNA ligase and a DNA / RNA deadenylase.

[1074] 315. The kit according to any one of aspects 210 to 314, wherein the kit further comprises reagents for a nucleic acid sequencing method.

[1075] 316. The kit of aspect 315, wherein the nucleic acid sequencing method is sequencing by synthesis, sequencing by ligation, sequencing by hybridization, polony sequencing, ion semiconductor sequencing, or pyrosequencing.

[1076] 317. The kit of aspect 315, wherein the nucleic acid sequencing method is single molecule real-time sequencing, nanopore-based sequencing, or direct imaging of DNA using advanced microscopy.

[1077] 318. The kit according to any one of aspects 210 to 317, wherein the kit further comprises reagents for amplifying the extended record tags.

[1078] 319. The kit according to any one of aspects 210-318, further comprising a reagent for adding a circulating marker.

[1079] 320. The kit of aspect 319, wherein the cycle labels provide information about the order in which the binding agent binds to the polypeptide.

[1080] 321. The kit according to aspect 319 or aspect 320, wherein a cycle marker is added to the encoding tag.

[1081] 322. The kit according to aspect 319 or aspect 320, wherein a cycle marker is added to the record label.

[1082] 323. The kit of aspect 319 or aspect 320, wherein a circulating label is added to the binding agent.

[1083] 324. The kit according to aspect 319 or aspect 320, wherein the circulating label is added independently of the coding label, the recording label and the binding agent.

[1084] 325. A kit according to any one of aspects 210 to 324, wherein the order of the encoded tag information contained in the extended record tags provides information about the binding order of the binding agents to the polypeptide.

[1085] 326. A kit according to any one of aspects 210 to 325, wherein the frequency of the encoded tag information contained in the extended record tag provides information about the binding frequency of the binding agent to the polypeptide.

[1086] 327. The kit according to any one of aspects 210 to 326, configured for analyzing one or more polypeptides from a sample comprising a plurality of protein complexes, proteins or polypeptides.

[1087] 328. The kit according to aspect 327 further comprises a means for partitioning multiple protein complexes, proteins or polypeptides within a sample into multiple compartments, wherein each compartment comprises multiple compartment labels, which are optionally attached to a support (e.g., a solid support), wherein the multiple compartment labels within a single compartment are the same and different from the compartment labels of other compartments.

[1088] 329. The kit of aspect 327 or 328, further comprising reagents for fragmenting a plurality of protein complexes, proteins and / or polypeptides into a plurality of polypeptides.

[1089] 330. The kit of aspect 328, wherein the compartment is a microfluidic droplet.

[1090] 331. The kit of aspect 328, wherein the compartment is a microwell.

[1091] 332. The kit of aspect 328, wherein the compartments are separate areas on a planar surface.

[1092] 333. The kit of any one of aspects 328 to 332, wherein each compartment contains, on average, a single cell.

[1093] 334. The kit according to any one of aspects 327 to 333, further comprising reagents for labeling a plurality of protein complexes, proteins or polypeptides with a plurality of universal DNA tags.

[1094] 335. A kit according to any one of aspects 328 to 334, wherein the reagents for transferring compartment label information to a record tag associated with the polypeptide comprise primer extension or ligation reagents.

[1095] 336. According to any one of aspects 328-335, 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.

[1096] 337. The kit according to any one of aspects 328 to 335, wherein the solid support comprises beads.

[1097] 338. The kit of aspect 337, 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.

[1098] 339. The kit according to any one of aspects 328 to 338, wherein the compartment labels comprise single-stranded or double-stranded nucleic acid molecules.

[1099] 340. The kit of any one of aspects 328-339, wherein the compartment labels comprise a barcode and, optionally, a UMI.

[1100] 341. The kit of aspect 340, wherein the solid support is a bead and the compartment label comprises a barcode, further wherein the bead comprising the plurality of compartment labels attached thereto is formed by split and pool synthesis.

[1101] 342. The kit of aspect 340, wherein the solid support is a bead and the compartment labels comprise barcodes, further wherein the bead comprising the plurality of compartment labels attached thereto is formed by a single synthesis or immobilization.

[1102] 343. The kit of any one of aspects 328-342, wherein the compartment label is a component of a record label, the record label optionally further comprising a spacer, a barcode sequence, a unique molecular identifier, a universal priming site, or any combination thereof.

[1103] 344. The kit according to any one of aspects 328 to 343, wherein the compartment tag further comprises a functional portion capable of reacting with an internal amino acid, a peptide backbone or an N-terminal amino acid of the plurality of protein complexes, proteins or polypeptides.

[1104] 345. The kit of aspect 344, wherein the functional moiety is an aldehyde, an azide / alkyne, or a maleimide / thiol, or an epoxide / nucleophile, or an inverse electron demand Diels-Alder (iEDDA) group.

[1105] 346. The kit of aspect 344, wherein the functional moiety is an aldehyde group.

[1106] 347. The kit according to any one of aspects 328 to 346, wherein the plurality of compartment labels are formed by printing, spotting, inkjet printing the compartment labels into the compartments, or a combination thereof.

[1107] 348. The kit of any one of aspects 328 to 347, wherein the compartment tag further comprises a polypeptide.

[1108] 349. The method of aspect 348, wherein the compartment tag polypeptide comprises a protein ligase recognition sequence.

[1109] 350. The kit of aspect 349, wherein the protein ligase is butelase I or a homologue thereof.

[1110] 351. The kit of any one of aspects 328 to 350, wherein the reagent for fragmenting the plurality of polypeptides comprises a protease.

[1111] 352. The kit of aspect 351, wherein the protease is a metalloprotease.

[1112] 353. The kit of aspect 352, further comprising an agent for modulating the activity of a metalloprotease, eg, an agent for photon-stimulated release of a metal cation of the metalloprotease.

[1113] 354. The kit according to any one of aspects 328 to 353, further comprising reagents for subtracting one or more abundant proteins from the sample prior to partitioning the plurality of polypeptides into the plurality of compartments.

[1114] 355. The kit according to any one of aspects 328 to 354, further comprising releasing the compartment tags from the solid support prior to attaching the plurality of peptides to the compartment tags.

[1115] 356. The kit of aspect 328, further comprising reagents for attaching the compartment-labeled polypeptide to a solid support associated with the record label.

[1116] 357. A method for screening polypeptide functionalization reagents, amino acid elimination reagents and / or reaction conditions, the method comprising the steps of:

[1117] (a) contacting the polynucleotide with a polypeptide functionalizing agent and / or an amino acid eliminating agent under reaction conditions; and

[1118] (b) evaluating the effect of step (a) on the polynucleotide, and optionally identifying polypeptide functionalization reagents, amino acid elimination reagents, and / or reaction conditions that have no or minimal effect on the polynucleotide.

[1119] 358. The method of aspect 357, wherein the polynucleotide comprises at least about 4 nucleotides.

[1120] 359. The method of aspect 357, wherein the polynucleotide comprises up to about 10 kb of nucleotides.

[1121] 360. The method of any one of claims 357-359, wherein the polynucleotide is a DNA polynucleotide.

[1122] 361. The method according to any one of aspects 357 to 360, wherein the polynucleotide is genomic DNA or the method is performed in the presence of genomic DNA.

[1123] 362. The method according to any one of aspects 357 to 360, wherein the polynucleotide is an isolated polynucleotide.

[1124] 363. The method according to any one of aspects 357 to 360, wherein the polynucleotide is part of a polypeptide binding agent.

[1125] 364. The method according to any one of aspects 357 to 363, wherein the polynucleotide is contacted with the polypeptide functionalising agent and / or the amino acid eliminating agent under reaction conditions in the absence of the polypeptide.

[1126] 365. The method according to any one of aspects 357 to 363, wherein the polynucleotide is contacted with a polypeptide functionalising agent and / or an amino acid eliminating agent under reaction conditions in the presence of the polypeptide.

[1127] 366. The method of aspect 365, wherein the polynucleotide is part of a polypeptide binding agent.

[1128] 367. The method according to any one of aspects 357 to 366, wherein the polypeptide functionalizing agent comprises a compound selected from the group consisting of:

[1129] (i) Compound of formula (I):

[1130]

[1131] or a salt or conjugate thereof,

[1132] in,

[1133] R 1 and R 2 Each independently is H, C1-6 Alkyl, cycloalkyl, -C(O)R a ,-C(O)OR b or -S(O)2R c ;

[1134] R a , R b and R c Each independently is H, C 1-6 Alkyl, C 1-6 haloalkyl, arylalkyl, aryl or heteroaryl, wherein C 1-6 Alkyl, C 1-6 haloalkyl, arylalkyl, aryl and heteroaryl are each unsubstituted or substituted;

[1135] R 3 is heteroaryl, -NR d C(O)OR e or –SR f , wherein heteroaryl is unsubstituted or substituted;

[1136] R d , R e and R f Each independently is H or C 1-6 alkyl; and

[1137] Optionally, when R 3 yes When R 1 and R 2 Not all are H;

[1138] (ii) Compounds of formula (II):

[1139]

[1140] or a salt or conjugate thereof,

[1141] in,

[1142] R 4 It is H, C 1-6 Alkyl, cycloalkyl, –C(O)R g OR –C(O)OR g ;and

[1143] R g H, C 1-6 Alkyl, C 2-6 Alkenyl, C 1-6 Halogenated alkyl or arylalkyl, wherein C 1-6 Alkyl, C 2-6 Alkenyl, C 1-6 Haloalkyl and arylalkyl are each unsubstituted or substituted;

[1144] (iii) Compounds of formula (III):

[1145] R 5 -N=C=S(III)

[1146] or a salt or conjugate thereof,

[1147] in,

[1148] R 5 It is C 1-6 Alkyl, C 2-6 alkenyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl;

[1149] Among them C 1-6 Alkyl, C 2-6 Alkenyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl are each unsubstituted or selected from halogen, -NR h R i , -S(O)2R j or one or more groups consisting of a heterocyclic group;

[1150] R h , R i and R j Each independently is H, C 1-6 Alkyl, C 1-6 haloalkyl, arylalkyl, aryl or heteroaryl, wherein C 1-6 Alkyl, C 1-6 Haloalkyl, arylalkyl, aryl and heteroaryl are each unsubstituted or substituted.

[1151] (iv) Compounds of formula (IV):

[1152]

[1153] or a salt or conjugate thereof,

[1154] in,

[1155] R 6 and R 7 Each independently is H, C 1-6 Alkyl, -CO2C 1-4 Alkyl, -OR k , aryl or cycloalkyl, wherein C 1-6 Alkyl, -CO2C 1-4 Alkyl, -OR k , aryl and cycloalkyl are each unsubstituted or substituted; and

[1156] R k H, C 1-6 Alkyl or heterocyclic group, wherein C1-6 The alkyl group and the heterocyclyl group are each unsubstituted or substituted;

[1157] (v) Compounds of formula (V):

[1158]

[1159] or a salt or conjugate thereof,

[1160] in,

[1161] R 8 Is a halogen or –OR m ;

[1162] R m H, C 1-6 an alkyl or heterocyclic group; and

[1163] R 9 is hydrogen, halogen or C 1-6 alkyl halide;

[1164] (vi) Metal complex of formula (VI):

[1165] ML n (VI)

[1166] or a salt or conjugate thereof,

[1167] in,

[1168] M is a metal selected from the group consisting of Co, Cu, Pd, Pt, Zn and Ni;

[1169] L is a ligand selected from the group consisting of: -OH, -OH2, 2,2'-bipyridine (bpy), 1,5-dithiocyclooctane (dtco), 1,2-bis(diphenylphosphino)ethane (dppe), ethylenediamine (en) and triethylenetetramine (trien); and

[1170] n is an integer from 1 to 8, inclusive;

[1171] wherein each L may be the same or different; and

[1172] (vii) Compounds of formula (VII):

[1173]

[1174] or a salt or conjugate thereof,

[1175] in,

[1176] G 1 It is N, NR 13 or CR 13 R14 ;

[1177] G 2 is N or CH;

[1178] p is 0 or 1;

[1179] R 10 , R 11 , R 12 , R 13 and R 14 Each independently selected from H, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 Alkylamine and C 1-6 Alkylhydroxylamine, where C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 Alkylamine and C 1-6 The alkylhydroxylamines are each unsubstituted or substituted, and R 10 and R 11 may optionally form a ring together; and

[1180] R 15 is H or OH.

[1181] 368. The method according to any one of aspects 357 to 367, wherein the amino acid elimination reagent is a chemical cleavage reagent or an enzymatic cleavage reagent.

[1182] 369. The method of aspect 368, wherein the amino acid elimination reagent is a carboxypeptidase or an aminopeptidase, or a variant, mutant, or modified protein thereof; a hydrolase, or a variant, mutant, or modified protein thereof; a mild Edman degradation reagent; an Edmanase enzyme; TFA; a base; or any combination thereof.

[1183] 370. The method according to any one of aspects 357-369, wherein the reaction conditions comprise reaction time, reaction temperature, reaction pH, solvent type, co-solvent, catalyst and ionic liquid, and electrochemical potential.

[1184] 371. The method of any one of aspects 357-370, wherein step (a) is performed in solution.

[1185] 372. The method of any one of aspects 357-370, wherein step (a) is performed in solid phase.

[1186] 373. A method according to any one of aspects 357 to 372, wherein the effect of step (a) on the polynucleotide is assessed by evaluating the presence, absence or amount of modification of the polynucleotide by the polypeptide functionalising agent, amino acid eliminating agent and / or reaction conditions.

[1187] 374. The method of any one of aspects 357 to 373, wherein less than 50% of the modifications in the polynucleotide recognize a polypeptide functionalizing agent, amino acid eliminating agent and / or reaction conditions that have no or minimal effect on the polynucleotide, compared to a corresponding polynucleotide not contacted with the polypeptide functionalizing agent and / or amino acid eliminating agent under the reaction conditions.

[1188] 375. A kit for screening polypeptide functionalizing agents, amino acid elimination agents and / or reaction conditions, comprising: (a) a polynucleotide; (b) a polypeptide functionalizing agent and / or an amino acid elimination agent; (c) a tool for evaluating the effects of polypeptide functionalizing agents, amino acid elimination agents and / or reaction conditions used for polypeptide functionalization or elimination on the polynucleotide.

[1189] 376. The kit of aspect 375, wherein the polypeptide functionalization reagent comprises a compound selected from the group consisting of:

[1190] (i) Compound of formula (I):

[1191]

[1192] or a salt or conjugate thereof,

[1193] in,

[1194] R 1 and R 2 Each independently is H, C 1-6 Alkyl, cycloalkyl, -C(O)R a ,-C(O)OR b or -S(O)2R c ;

[1195] R a , R b and R c Each independently is H, C 1-6 Alkyl, C 1-6 haloalkyl, arylalkyl, aryl or heteroaryl, wherein C 1-6 Alkyl, C 1-6 haloalkyl, arylalkyl, aryl and heteroaryl are each unsubstituted or substituted;

[1196] R 3 is heteroaryl, -NR d C(O)OR e or –SR f , wherein heteroaryl is unsubstituted or substituted;

[1197] R d , R e and Rf Each independently is H or C 1-6 alkyl; and

[1198] Optionally, when R 3 yes When R 1 and R 2 Not all are H;

[1199] (ii) Compounds of formula (II):

[1200]

[1201] or a salt or conjugate thereof,

[1202] in,

[1203] R 4 It is H, C 1-6 Alkyl, cycloalkyl, –C(O)R g OR –C(O)OR g ;and

[1204] R g H, C 1-6 Alkyl, C 2-6 Alkenyl, C 1-6 Halogenated alkyl or arylalkyl, wherein C 1-6 Alkyl, C 2-6 Alkenyl, C 1-6 Haloalkyl and arylalkyl are each unsubstituted or substituted;

[1205] (iii) Compounds of formula (III):

[1206] R 5 -N=C=S (III)

[1207] or a salt or conjugate thereof,

[1208] in,

[1209] R 5 It is C 1-6 Alkyl, C 2-6 alkenyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl;

[1210] Among them C 1-6 Alkyl, C 2-6 Alkenyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl are each unsubstituted or selected from halogen, -NR h R i , -S(O)2R j or one or more groups consisting of a heterocyclic group;

[1211] R h, R i and R j Each independently is H, C 1-6 Alkyl, C 1-6 haloalkyl, arylalkyl, aryl or heteroaryl, wherein C 1-6 Alkyl, C 1-6 Haloalkyl, arylalkyl, aryl and heteroaryl are each unsubstituted or substituted.

[1212] (iv) Compounds of formula (IV):

[1213]

[1214] or a salt or conjugate thereof,

[1215] in,

[1216] R 6 and R 7 Each independently is H, C 1-6 Alkyl, -CO2C 1-4 Alkyl, -OR k , aryl or cycloalkyl, wherein C 1-6 Alkyl, -CO2C 1-4 Alkyl, -OR k , aryl and cycloalkyl are each unsubstituted or substituted; and

[1217] R k H, C 1-6 Alkyl or heterocyclic group, wherein C 1-6 The alkyl group and the heterocyclyl group are each unsubstituted or substituted;

[1218] (v) Compounds of formula (V):

[1219]

[1220] or a salt or conjugate thereof,

[1221] in,

[1222] R 8 Is a halogen or –OR m ;

[1223] R m H, C 1-6 an alkyl or heterocyclic group; and

[1224] R 9 is hydrogen, halogen or C 1-6 alkyl halide;

[1225] (vi) Metal complex of formula (VI):

[1226] ML n(VI)

[1227] or a salt or conjugate thereof,

[1228] in,

[1229] M is a metal selected from the group consisting of Co, Cu, Pd, Pt, Zn and Ni;

[1230] L is a ligand selected from the group consisting of: -OH, -OH2, 2,2'-bipyridine (bpy), 1,5-dithiocyclooctane (dtco), 1,2-bis(diphenylphosphino)ethane (dppe), ethylenediamine (en) and triethylenetetramine (trien); and

[1231] n is an integer from 1 to 8, inclusive;

[1232] wherein each L may be the same or different; and

[1233] (vii) Compounds of formula (VII):

[1234]

[1235] or a salt or conjugate thereof,

[1236] in,

[1237] G 1 It is N, NR 13 or CR 13 R 14 ;

[1238] G 2 is N or CH;

[1239] p is 0 or 1;

[1240] R 10 , R 11 , R 12 , R 13 and R 14 Each independently selected from H, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 Alkylamine and C 1-6 Alkylhydroxylamine, where C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 Alkylamine and C 1-6 The alkylhydroxylamines are each unsubstituted or substituted, and R 10 and R 11 may optionally form a ring together; and

[1241] R 15 is H or OH.

[1242] 377. The kit of aspect 375 or aspect 376, comprising a polypeptide functionalisation reagent and an amino acid elimination reagent.

[1243] 378. A method for sequencing a polypeptide, the method comprising:

[1244] (a) attaching the polypeptide to a support or substrate, or providing the polypeptide in a solution;

[1245] (b) functionalizing the N-terminal amino acid (NTAA) of the polypeptide with a chemical agent, wherein the chemical agent comprises a compound selected from the group consisting of

[1246] (i) Compound of formula (I):

[1247]

[1248] or a salt or conjugate thereof,

[1249] in,

[1250] R 1 and R 2 Each independently is H, C 1-6 Alkyl, cycloalkyl, -C(O)R a ,-C(O)OR b or -S(O)2R c ;

[1251] R a , R b and R c Each independently is H, C 1-6 Alkyl, C 1-6 haloalkyl, arylalkyl, aryl or heteroaryl, wherein C 1-6 Alkyl, C 1-6 haloalkyl, arylalkyl, aryl and heteroaryl are each unsubstituted or substituted;

[1252] R 3 is heteroaryl, -NR d C(O)OR e or –SR f , wherein heteroaryl is unsubstituted or substituted;

[1253] R d , R e and R f Each independently is H or C 1-6 alkyl; and

[1254] Optionally, when R 3 yes When R 1and R 2 Not all are H;

[1255] (ii) Compounds of formula (II):

[1256]

[1257] or a salt or conjugate thereof,

[1258] in,

[1259] R 4 It is H, C 1-6 Alkyl, cycloalkyl, –C(O)R g OR –C(O)OR g ;and

[1260] R g H, C 1-6 Alkyl, C 2-6 Alkenyl, C 1-6 Halogenated alkyl or arylalkyl, wherein C 1-6 Alkyl, C 2-6 Alkenyl, C 1-6 Haloalkyl and arylalkyl are each unsubstituted or substituted;

[1261] (iii) Compounds of formula (III):

[1262] R 5 -N=C=S (iii)

[1263] or a salt or conjugate thereof,

[1264] in,

[1265] R 5 It is C 1-6 Alkyl, C 2-6 alkenyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl;

[1266] Among them C 1-6 Alkyl, C 2-6 Alkenyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl are each unsubstituted or selected from halogen, -NR h R i , -S(O)2R j or one or more groups consisting of a heterocyclic group;

[1267] R h , R i and R j Each independently is H, C 1-6 Alkyl, C 1-6 haloalkyl, arylalkyl, aryl or heteroaryl, wherein C 1-6 Alkyl, C1-6 Haloalkyl, arylalkyl, aryl and heteroaryl are each unsubstituted or substituted.

[1268] (iv) Compounds of formula (IV):

[1269]

[1270] or a salt or conjugate thereof,

[1271] in,

[1272] R 6 and R 7 Each independently is H, C 1-6 Alkyl, -CO2C 1-4 Alkyl, -OR k , aryl or cycloalkyl, wherein C 1-6 Alkyl, -CO2C 1-4 Alkyl, -OR k , aryl and cycloalkyl are each unsubstituted or substituted; and

[1273] R k H, C 1-6 Alkyl or heterocyclic group, wherein C 1-6 The alkyl group and the heterocyclyl group are each unsubstituted or substituted;

[1274] (v) Compounds of formula (V):

[1275]

[1276] or a salt or conjugate thereof,

[1277] in,

[1278] R 8 Is a halogen or –OR m ;

[1279] R m H, C 1-6 an alkyl or heterocyclic group; and

[1280] R 9 is hydrogen, halogen or C 1-6 alkyl halide;

[1281] (vi) Metal complex of formula (VI):

[1282] ML n (VI)

[1283] or a salt or conjugate thereof,

[1284] in,

[1285] M is a metal selected from the group consisting of Co, Cu, Pd, Pt, Zn and Ni;

[1286] L is a ligand selected from the group consisting of: -OH, -OH2, 2,2'-bipyridine (bpy), 1,5-dithiocyclooctane (dtco), 1,2-bis(diphenylphosphino)ethane (dppe), ethylenediamine (en) and triethylenetetramine (trien); and

[1287] n is an integer from 1 to 8, inclusive;

[1288] wherein each L may be the same or different; and

[1289] (vii) Compounds of formula (VII):

[1290]

[1291] or a salt or conjugate thereof,

[1292] in,

[1293] G 1 It is N, NR 13 or CR 13 R 14 ;

[1294] G 2 is N or CH;

[1295] p is 0 or 1;

[1296] R 10 , R 11 , R 12 , R 13 and R 14 Each independently selected from H, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 Alkylamine and C 1-6 Alkylhydroxylamine, where C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 Alkylamine and C 1-6 The alkylhydroxylamines are each unsubstituted or substituted, and R 10 and R 11 may optionally form a ring together; and

[1297] R 15 is H or OH;

[1298] (c) contacting the polypeptide with a plurality of binding agents, each of which comprises a binding moiety capable of binding to the functionalized NTAA and a detectable label;

[1299] (d) detecting the detectable label of the binding agent bound to the polypeptide, thereby identifying the N-terminal amino acid of the polypeptide;

[1300] (e) eliminating the functionalized NTAA to expose a new NTAA; and

[1301] (f) repeating steps (b)-(d) to determine the sequence of at least a portion of the polypeptide.

[1302] 379. The method of aspect 378, wherein step (b) is performed before step (c).

[1303] 380. The method of aspect 378, wherein step (b) is performed after step (c) and before step (d).

[1304] 381. The method of aspect 378, wherein step (b) is performed after step (c) and step (d).

[1305] 382. The method according to any one of aspects 378 to 381, wherein the polypeptide is obtained by fragmenting a protein from a biological sample.

[1306] 383. The method of any one of aspects 378-382, wherein the 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 wafer chip, a flow-through chip, a biochip comprising 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.

[1307] 384. The method according to any one of aspects 378 to 383, wherein NTAA is removed from the polypeptide by chemical cleavage or enzymatic cleavage.

[1308] 385. The method of aspects 378-384, wherein NTAA is eliminated by a carboxypeptidase or an aminopeptidase, or a variant, mutant or modified protein thereof; a hydrolase, or a variant, mutant or modified protein thereof; mild Edman degradation; an Edmanase enzyme; TFA, alkali; or any combination thereof.

[1309] 386. The method of any one of aspects 378 to 385, wherein the polypeptide is covalently attached to the support or substrate.

[1310] 387. The method of any one of aspects 378-386, wherein the support or substrate is optically transparent.

[1311] 388. The method according to any one of aspects 378 to 387, wherein the support or substrate comprises a plurality of spatially resolved attachment points, and step a) comprises attaching the polypeptide to the spatially resolved attachment points.

[1312] 389. The method according to any one of aspects 378 to 388, wherein the binding portion of the binding agent comprises a peptide or protein.

[1313] 390. The method of any one of aspects 378-389, wherein the binding portion of the binding agent comprises an aminopeptidase or a variant, mutant or modified protein thereof; an aminoacyl-tRNA synthetase or a variant, mutant or modified protein thereof; an anticalin or a variant, mutant or modified protein thereof; a Clps (e.g., ClpS2) or a variant, mutant or modified protein thereof; a UBR box protein or a variant, mutant or modified protein thereof; or a modified small molecule that binds an amino acid, i.e., vancomycin or a variant, mutant or modified molecule thereof; or an antibody or a binding fragment thereof; or any combination thereof.

[1314] 391. The method of any one of aspects 378-390, wherein the chemical agent comprises a conjugate selected from the group consisting of:

[1315]

[1316] where R 1 , R 2 and R 3 As defined for formula (I) in aspect 1, and Q is a ligand;

[1317]

[1318] where R 4 As defined for formula (II) in aspect 1, and Q is a ligand;

[1319]

[1320] where R 5 As defined for formula (III) in aspect 1, and Q is a ligand;

[1321]

[1322] where R 6 and R 7 As defined for formula (IV) in aspect 1, and Q is a ligand;

[1323]

[1324] where R 8 and R9 As defined for formula (V) in aspect 1, and Q is a ligand;

[1325] (ML n )-Q formula (VI)-Q,

[1326] wherein M, L and N are as defined for formula (V) in aspect 1, and Q is a ligand;

[1327]

[1328] where R 10 , R 11 , R 12 , R 15 , G 1 , G 2 and p are as defined in Aspect 1 for Formula (VII), and Q is a ligand.

[1329] 392. The method of any one of aspects 378 to 391, wherein step (b) comprises functionalizing the NTAA with a second chemical agent selected from the group consisting of formula (VIIIa) and (VIIIb):

[1330]

[1331] or a salt or conjugate thereof,

[1332] in,

[1333] R 13 It is H, C 1-6 Alkyl, aryl, heteroaryl, cycloalkyl or heterocycloalkyl, wherein C 1-6 Alkyl, aryl, heteroaryl, cycloalkyl and heterocycloalkyl are each unsubstituted or substituted; or

[1334] R 13 -X (VIIIb)

[1335] in,

[1336] R 13 C 1-6 alkyl, aryl, heteroaryl, cycloalkyl or heterocycloalkyl, each of which is unsubstituted or substituted; and

[1337] X is a halogen.

[1338] 393. The method of any one of aspects 378 to 392, wherein the polypeptide is a partially or fully digested protein.

[1339] 394. A method for sequencing a plurality of polypeptide molecules in a sample, the method comprising:

[1340] (a) attaching polypeptide molecules in a sample to multiple spatially resolved attachment sites on a support or substrate;

[1341] (b) functionalizing the N-terminal amino acid (NTAA) of the polypeptide molecule with a chemical reagent, wherein the chemical reagent comprises a compound selected from the group consisting of:

[1342] (i) Compound of formula (I):

[1343]

[1344] or a salt or conjugate thereof,

[1345] in,

[1346] R 1 and R 2 Each independently is H, C 1-6 Alkyl, cycloalkyl, -C(O)R a ,-C(O)OR b or -S(O)2R c ;

[1347] R a , R b and R c Each independently is H, C 1-6 Alkyl, C 1-6 haloalkyl, arylalkyl, aryl or heteroaryl, wherein C 1-6 Alkyl, C 1-6 haloalkyl, arylalkyl, aryl and heteroaryl are each unsubstituted or substituted;

[1348] R 3 is heteroaryl, -NR d C(O)OR e or –SR f , wherein heteroaryl is unsubstituted or substituted;

[1349] R d , R e and R f Each independently is H or C 1-6 alkyl; and

[1350] Optionally, when R 3 yes When R 1 and R 2 Not all are H;

[1351] (ii) Compounds of formula (II):

[1352]

[1353] or a salt or conjugate thereof,

[1354] in,

[1355] R 4 It is H, C 1-6 Alkyl, cycloalkyl, –C(O)R g OR –C(O)OR g ;and

[1356] R g H, C 1-6 Alkyl, C 2-6 Alkenyl, C 1-6 Halogenated alkyl or arylalkyl, wherein C 1-6 Alkyl, C 2-6 Alkenyl, C 1-6 Haloalkyl and arylalkyl are each unsubstituted or substituted;

[1357] (iii) Compounds of formula (III):

[1358] R 5 -N=C=S (iii)

[1359] or a salt or conjugate thereof,

[1360] in,

[1361] R 5 It is C 1-6 Alkyl, C 2-6 alkenyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl;

[1362] Among them C 1-6 Alkyl, C 2-6 Alkenyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl are each unsubstituted or selected from halogen, -NR h R i , -S(O)2R j or one or more groups consisting of a heterocyclic group;

[1363] R h , R i and R j Each independently is H, C 1-6 Alkyl, C 1-6 haloalkyl, arylalkyl, aryl or heteroaryl, wherein C 1-6 Alkyl, C 1-6 Haloalkyl, arylalkyl, aryl and heteroaryl are each unsubstituted or substituted.

[1364] (iv) Compounds of formula (IV):

[1365]

[1366] or a salt or conjugate thereof,

[1367] in,

[1368] R 6 and R 7 Each independently is H, C 1-6 Alkyl, -CO2C 1-4 Alkyl, -OR k , aryl or cycloalkyl, wherein C 1-6 Alkyl, -CO2C 1-4 Alkyl, -OR k , aryl and cycloalkyl are each unsubstituted or substituted; and

[1369] R k H, C 1-6 Alkyl or heterocyclic group, wherein C 1-6 The alkyl group and the heterocyclyl group are each unsubstituted or substituted;

[1370] (v) Compounds of formula (V):

[1371]

[1372] or a salt or conjugate thereof,

[1373] in,

[1374] R 8 Is a halogen or –OR m ;

[1375] R m H, C 1-6 an alkyl or heterocyclic group; and

[1376] R 9 is hydrogen, halogen or C 1-6 alkyl halide;

[1377] (vi) Metal complex of formula (VI):

[1378] ML n (VI)

[1379] or a salt or conjugate thereof,

[1380] in,

[1381] M is a metal selected from the group consisting of Co, Cu, Pd, Pt, Zn and Ni;

[1382] L is a ligand selected from the group consisting of: -OH, -OH2, 2,2'-bipyridine (bpy), 1,5-dithiocyclooctane (dtco), 1,2-bis(diphenylphosphino)ethane (dppe), ethylenediamine (en) and triethylenetetramine (trien); and

[1383] n is an integer from 1 to 8, inclusive;

[1384] wherein each L may be the same or different; and

[1385] (vii) Compounds of formula (VII):

[1386]

[1387] or a salt or conjugate thereof,

[1388] in,

[1389] G 1 It is N, NR 13 or CR 13 R 14 ;

[1390] G 2 is N or CH;

[1391] p is 0 or 1;

[1392] R 10 , R 11 , R 12 , R 13 and R 14 Each independently selected from H, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 Alkylamine and C 1-6 Alkylhydroxylamine, where C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 Alkylamine and C 1-6 The alkylhydroxylamines are each unsubstituted or substituted, and R 10 and R 11 may optionally form a ring together; and

[1393] R 15 is H or OH;

[1394] (c) contacting the polypeptide with a plurality of binding agents, each of which comprises a binding moiety capable of binding to the functionalized NTAA and a detectable label;

[1395] (d) optically detecting the fluorescent label of the probe bound to each of the plurality of polypeptide molecules that are spatially resolved and attached to a support or substrate;

[1396] (e) eliminating the functionalized NTAA from each polypeptide; and

[1397] (f) repeating steps b) through d) to determine the sequence of at least a portion of one or more of the plurality of polypeptide molecules that is spatially resolved and attached to the support or substrate.

[1398] 395. The method of aspect 394, wherein step (b) is performed before step (c).

[1399] 396. The method of aspect 394, wherein step (b) is performed after step (c) and before step (d).

[1400] 397. The method of aspect 394, wherein step (b) is performed after step (c) and step (d).

[1401] 398. The method of any one of aspects 394-397, wherein the sample comprises a biological fluid, a cell extract, or a tissue extract.

[1402] 399. The method according to any one of aspects 394 to 398, further comprising comparing the sequence of the at least one polypeptide molecule determined in step e) to a reference protein sequence database.

[1403] 400. The method according to any one of aspects 394 to 399, further comprising comparing the sequence of each polypeptide determined in step e), grouping similar polypeptide sequences and counting the number of instances of each similar polypeptide sequence.

[1404] 401. The method of any one of aspects 394-400, wherein the fluorescent label is a fluorescent group, a color-coded nanoparticle, or a quantum dot.

[1405] 402. A kit for sequencing a polypeptide, comprising:

[1406] (a) reagents for attaching the polypeptide to a support or substrate, or reagents for providing the polypeptide in solution;

[1407] (b) a reagent for functionalizing the N-terminal amino acid (NTAA) of a polypeptide, wherein the reagent comprises a compound selected from the group consisting of:

[1408] (i) Compound of formula (I):

[1409]

[1410] or a salt or conjugate thereof,

[1411] in,

[1412] R 1 and R 2 Each independently is H, C 1-6 Alkyl, cycloalkyl, -C(O)R a ,-C(O)OR b or -S(O)2R c ;

[1413] R a , R b and R c Each independently is H, C 1-6 Alkyl, C 1-6 haloalkyl, arylalkyl, aryl or heteroaryl, wherein C 1-6 Alkyl, C 1-6 haloalkyl, arylalkyl, aryl and heteroaryl are each unsubstituted or substituted;

[1414] R 3 is heteroaryl, -NR d C(O)OR e or –SR f , wherein heteroaryl is unsubstituted or substituted;

[1415] R d , R e and R f Each independently is H or C 1-6 alkyl; and

[1416] Optionally, when R 3 yes When R 1 and R 2 Not all are H;

[1417] (ii) Compounds of formula (II):

[1418]

[1419] or a salt or conjugate thereof,

[1420] in,

[1421] R 4 It is H, C 1-6 Alkyl, cycloalkyl, –C(O)R g OR –C(O)OR g ;and

[1422] R g H, C 1-6 Alkyl, C 2-6 Alkenyl, C 1-6 Halogenated alkyl or arylalkyl, wherein C 1-6 Alkyl, C 2-6Alkenyl, C 1-6 Haloalkyl and arylalkyl are each unsubstituted or substituted;

[1423] (iii) Compounds of formula (III):

[1424] R 5 -N=C=S (iii)

[1425] or a salt or conjugate thereof,

[1426] in,

[1427] R 5 It is C 1-6 Alkyl, C 2-6 alkenyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl;

[1428] Among them C 1-6 Alkyl, C 2-6 Alkenyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl are each unsubstituted or selected from halogen, -NR h R i , -S(O)2R j or one or more groups consisting of a heterocyclic group;

[1429] R h , R i and R j Each independently is H, C 1-6 Alkyl, C 1-6 haloalkyl, arylalkyl, aryl or heteroaryl, wherein C 1-6 Alkyl, C 1-6 Haloalkyl, arylalkyl, aryl and heteroaryl are each unsubstituted or substituted.

[1430] (iv) Compounds of formula (IV):

[1431]

[1432] or a salt or conjugate thereof,

[1433] in,

[1434] R 6 and R 7 Each independently is H, C 1-6 Alkyl, -CO2C 1-4 Alkyl, -OR k , aryl or cycloalkyl, wherein C 1-6 Alkyl, -CO2C 1-4 Alkyl, -OR k , aryl and cycloalkyl are each unsubstituted or substituted; and

[1435] R k H, C 1-6 Alkyl or heterocyclic group, wherein C 1-6 The alkyl group and the heterocyclyl group are each unsubstituted or substituted;

[1436] (v) Compounds of formula (V):

[1437]

[1438] or a salt or conjugate thereof,

[1439] in,

[1440] R 8 Is a halogen or –OR m ;

[1441] R m H, C 1-6 an alkyl or heterocyclic group; and

[1442] R 9 is hydrogen, halogen or C 1-6 alkyl halide;

[1443] (vi) Metal complex of formula (VI):

[1444] ML n (VI)

[1445] or a salt or conjugate thereof,

[1446] in,

[1447] M is a metal selected from the group consisting of Co, Cu, Pd, Pt, Zn and Ni;

[1448] L is a ligand selected from the group consisting of: -OH, -OH2, 2,2'-bipyridine (bpy), 1,5-dithiocyclooctane (dtco), 1,2-bis(diphenylphosphino)ethane (dppe), ethylenediamine (en) and triethylenetetramine (trien); and

[1449] n is an integer from 1 to 8, inclusive;

[1450] wherein each L may be the same or different; and

[1451] (vii) Compounds of formula (VII):

[1452]

[1453] or a salt or conjugate thereof,

[1454] in,

[1455] G 1It is N, NR 13 or CR 13 R 14 ;

[1456] G 2 is N or CH;

[1457] p is 0 or 1;

[1458] R 10 , R 11 , R 12 , R 13 and R 14 Each independently selected from H, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 Alkylamine and C 1-6 Alkylhydroxylamine, where C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 Alkylamine and C 1-6 The alkylhydroxylamines are each unsubstituted or substituted, and R 10 and R 11 may optionally form a ring together; and

[1459] R 15 is H or OH; and

[1460] (c) A binding agent comprising a binding moiety capable of binding to functionalized NTAA and a detectable label.

[1461] 403. The kit of aspect 402, wherein the kit further comprises reagents for eliminating functionalized NTAAs to expose new NTAAs.

[1462] 404. The kit according to aspect 402 or aspect 403, wherein the peptides are obtained by fragmenting proteins from a biological sample.

[1463] 405. According to the kit of any one of aspects 402-404, the support or substrate 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.

[1464] 406. The kit of aspects 403-405, wherein the reagent for eliminating functionalized NTAA is a carboxypeptidase or an aminopeptidase or a variant, mutant or modified protein thereof; a hydrolase or a variant, mutant or modified protein thereof; a mild Edman degradation reagent; an Edmanase enzyme; TFA; a base; or any combination thereof.

[1465] 407. The kit according to any one of aspects 402-406, wherein the polypeptide is covalently attached to the support or substrate.

[1466] 408. The kit according to any one of aspects 402-407, wherein the support or substrate is optically transparent.

[1467] 409. The kit according to any one of aspects 402 to 408, wherein the support or substrate comprises a plurality of spatially resolved attachment points, and step a) comprises attaching the polypeptide to the spatially resolved attachment points.

[1468] 410. The kit according to any one of aspects 402 to 409, wherein the binding moiety of the binding agent comprises a peptide or a protein.

[1469] 411. The kit of any one of aspects 402-410, wherein the binding portion of the binding agent comprises an aminopeptidase or a variant, mutant or modified protein thereof; an aminoacyl-tRNA synthetase or a variant, mutant or modified protein thereof; an anticalin or a variant, mutant or modified protein thereof; a Clps (e.g., ClpS2) or a variant, mutant or modified protein thereof; a UBR box protein or a variant, mutant or modified protein thereof; or a modified small molecule that binds an amino acid, i.e., vancomycin or a variant, mutant or modified molecule thereof; or an antibody or binding fragment thereof; or any combination thereof.

[1470] 412. The kit according to any one of aspects 402-411, wherein the chemical reagent comprises a conjugate selected from the group consisting of:

[1471]

[1472] where R 1 , R 2 and R 3 As defined for formula (I) in aspect 1, and Q is a ligand;

[1473]

[1474] where R 4 As defined for formula (II) in aspect 1, and Q is a ligand;

[1475]

[1476] where R 5 As defined for formula (III) in aspect 1, and Q is a ligand;

[1477]

[1478] where R 6 and R 7 As defined for formula (IV) in aspect 1, and Q is a ligand;

[1479]

[1480] where R 8 and R 9 As defined for formula (V) in aspect 1, and Q is a ligand;

[1481] (ML n )-Q formula (VI)-Q,

[1482] wherein M, L and N are as defined for formula (V) in aspect 1, and Q is a ligand;

[1483]

[1484] where R 10 , R 11 , R 12 , R 15 , G 1 , G 2 and p are as defined in Aspect 1 for Formula (VII), and Q is a ligand.

[1485] Aspect 413. The kit according to any one of aspects 402-412, comprising a second chemical reagent selected from the group consisting of formula (VIIIa) and (VIIIb):

[1486]

[1487] or a salt or conjugate thereof,

[1488] in,

[1489] R 13 It is H, C 1-6 Alkyl, aryl, heteroaryl, cycloalkyl or heterocycloalkyl, wherein C 1-6 Alkyl, aryl, heteroaryl, cycloalkyl and heterocycloalkyl are each unsubstituted or substituted; or

[1490] R 13 -XX (VIIIb)

[1491] in,

[1492] R 13 C 1-6 alkyl, aryl, heteroaryl, cycloalkyl or heterocycloalkyl, each of which is unsubstituted or substituted; and

[1493] X is a halogen.

[1494] 414. The method according to any one of aspects 402-413, wherein the polypeptide is a partially or fully digested protein.

[1495] 415. A kit for sequencing a plurality of polypeptides, comprising:

[1496] (a) Reagents for attaching polypeptide molecules in a sample to a plurality of spatially resolved attachment sites on a support or substrate;

[1497] (b) a reagent for functionalizing the N-terminal amino acid (NTAA) of a polypeptide molecule, wherein the reagent comprises a compound selected from the group consisting of:

[1498] (i) Compound of formula (I):

[1499]

[1500] or a salt or conjugate thereof,

[1501] in,

[1502] R 1 and R 2 Each independently is H, C 1-6 Alkyl, cycloalkyl, -C(O)R a ,-C(O)OR b or -S(O)2R c ;

[1503] R a , R b and R c Each independently is H, C 1-6 Alkyl, C 1-6 haloalkyl, arylalkyl, aryl or heteroaryl, wherein C 1-6 Alkyl, C 1-6 haloalkyl, arylalkyl, aryl and heteroaryl are each unsubstituted or substituted;

[1504] R 3 is heteroaryl, -NR d C(O)OR e or –SR f , wherein heteroaryl is unsubstituted or substituted;

[1505] R d , R e and Rf Each independently is H or C 1-6 alkyl; and

[1506] Optionally, when R 3 yes When R 1 and R 2 Not all are H;

[1507] (ii) Compounds of formula (II):

[1508]

[1509] or a salt or conjugate thereof,

[1510] in,

[1511] R 4 It is H, C 1-6 Alkyl, cycloalkyl, –C(O)R g OR –C(O)OR g ;and

[1512] R g H, C 1-6 Alkyl, C 2-6 Alkenyl, C 1-6 Halogenated alkyl or arylalkyl, wherein C 1-6 Alkyl, C 2-6 Alkenyl, C 1-6 Haloalkyl and arylalkyl are each unsubstituted or substituted;

[1513] (iii) Compounds of formula (III):

[1514] R 5 -N=C=S (iii)

[1515] or a salt or conjugate thereof,

[1516] in,

[1517] R 5 It is C 1-6 Alkyl, C 2-6 alkenyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl;

[1518] Among them C 1-6 Alkyl, C 2-6 Alkenyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl are each unsubstituted or selected from halogen, -NR h R i , -S(O)2R j or one or more groups consisting of a heterocyclic group;

[1519] R h, R i and R j Each independently is H, C 1-6 Alkyl, C 1-6 haloalkyl, arylalkyl, aryl or heteroaryl, wherein C 1-6 Alkyl, C 1-6 Haloalkyl, arylalkyl, aryl and heteroaryl are each unsubstituted or substituted.

[1520] (iv) Compounds of formula (IV):

[1521]

[1522] or a salt or conjugate thereof,

[1523] in,

[1524] R 6 and R 7 Each independently is H, C 1-6 Alkyl, -CO2C 1-4 Alkyl, -OR k , aryl or cycloalkyl, wherein C 1-6 Alkyl, -CO2C 1-4 Alkyl, -OR k , aryl and cycloalkyl are each unsubstituted or substituted; and

[1525] R k H, C 1-6 Alkyl or heterocyclic group, wherein C 1-6 The alkyl group and the heterocyclyl group are each unsubstituted or substituted;

[1526] (v) Compounds of formula (V):

[1527]

[1528] or a salt or conjugate thereof,

[1529] in,

[1530] R 8 Is a halogen or –OR m ;

[1531] R m H, C 1-6 an alkyl or heterocyclic group; and

[1532] R 9 is hydrogen, halogen or C 1-6 alkyl halide;

[1533] (vi) Metal complex of formula (VI):

[1534] ML n(VI)

[1535] or a salt or conjugate thereof,

[1536] in,

[1537] M is a metal selected from the group consisting of Co, Cu, Pd, Pt, Zn and Ni;

[1538] L is a ligand selected from the group consisting of: -OH, -OH2, 2,2'-bipyridine (bpy), 1,5-dithiocyclooctane (dtco), 1,2-bis(diphenylphosphino)ethane (dppe), ethylenediamine (en) and triethylenetetramine (trien); and

[1539] n is an integer from 1 to 8, inclusive;

[1540] wherein each L may be the same or different; and

[1541] (vii) Compounds of formula (VII):

[1542]

[1543] or a salt or conjugate thereof,

[1544] in,

[1545] G 1 It is N, NR 13 or CR 13 R 14 ;

[1546] G 2 is N or CH;

[1547] p is 0 or 1;

[1548] R 10 , R 11 , R 12 , R 13 and R 14 Each independently selected from H, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 Alkylamine and C 1-6 Alkylhydroxylamine, where C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 Alkylamine and C 1-6 The alkylhydroxylamines are each unsubstituted or substituted, and R 10 and R 11 may optionally form a ring together; and

[1549] R 15is H or OH; and

[1550] (c) A binding agent comprising a binding moiety capable of binding to functionalized NTAA and a detectable label.

[1551] 416. The kit of aspect 415, wherein the kit further comprises reagents for eliminating functionalized NTAAs to expose new NTAAs.

[1552] Aspect 417. The kit according to aspect 415 or 416, wherein the sample comprises a biological fluid, a cell extract, or a tissue extract.

[1553] Aspect 418. The kit according to any one of aspects 415 to 417, wherein the fluorescent label is a fluorescent group, a color-coded nanoparticle or a quantum dot.

[1554] In any of the foregoing embodiments, the recording tag and / or encoding tag can be a small molecule, or a subunit, monomer, residue or structural unit of a sequenceable polymer (eg, a polynucleotide or a non-nucleic acid sequenceable polymer).

[1555] In any of the foregoing embodiments, the recording tag and / or the encoding tag may be a sequenceable polymer, such as a polynucleotide or a non-nucleic acid sequenceable polymer.

[1556] In any of the foregoing embodiments, the recording tag and / or encoding tag may comprise a small molecule, or a subunit, monomer, residue or building block of a sequenceable polymer (eg, a polynucleotide or a non-nucleic acid sequenceable polymer).

[1557] In any of the foregoing embodiments, the coding tag can be a small molecule, or a subunit, monomer, residue, or structural unit of a sequenceable polymer (e.g., a polynucleotide or a non-nucleic acid sequenceable polymer), and the recording tag is a sequenceable polymer. In some embodiments, the coding tag (small molecule, subunit, or monomer, etc.) can be added to the recording tag to form a sequenceable polymer, such as beads (e.g., various coding tags) on a string (e.g., an extended recording tag).

[1558] In any of the foregoing embodiments, the extended recording tag and / or the extended encoding tag can be a sequenceable polymer, such as a polynucleotide or a non-nucleic acid sequenceable polymer.

[1559] In one aspect, disclosed herein is a method comprising: (a) contacting an analyte with a first binding agent capable of binding to the analyte, wherein the first binding agent comprises a first coding portion having identification information about the first binding agent, the analyte is associated with a recording portion attached to a support, and the analyte and / or the recording portion is fixed to the support; (b) transferring the information of the first coding portion to a recording polymer to produce a first sequence-extended recording portion; (c) contacting the analyte with a second binding agent capable of binding to the analyte, wherein the second binding agent comprises a second coding portion having identification information about the second binding agent; (d) transferring the information of the second coding portion to the first sequence-extended recording portion to produce a second sequence-extended recording portion; and (e) analyzing the second sequence-extended recording portion. In one embodiment, the method further comprises the following steps between steps (d) and (e): (x) repeating steps (c) and (d) one or more times by replacing the second binding agent with a third (or higher order) binding agent capable of binding to the modified analyte, wherein the third (or higher order) binding agent comprises a third (or higher order) coding portion having identification information about the third (or higher order) binding agent; and (y) transferring the information of the third (or higher order) coding portion to the second (or higher order) extended record portion to produce a third (or higher order) extended record portion; and analyzing the third (or higher order) extended record portion in step (e).

[1560] In any of the foregoing embodiments, the recording portion can be a small molecule, or a subunit, monomer, residue or building block of a sequenceable polymer (eg, a polynucleotide or a non-nucleic acid sequenceable polymer).

[1561] In any of the foregoing embodiments, the coding portion can be a sequenceable polymer, such as a polynucleotide or a non-nucleic acid sequenceable polymer. In some embodiments, the first, second, third, and / or higher order coding portions comprise small molecules and / or subunits, monomers, residues, or building blocks of a sequenceable polymer (e.g., a polynucleotide or a non-nucleic acid sequenceable polymer).

[1562] In any of the foregoing embodiments, the first, second, third and / or higher order coding portion can be a small molecule, or a subunit, monomer, residue or building block of a sequenceable polymer (e.g., a polynucleotide or a non-nucleic acid sequenceable polymer).

[1563] In any of the foregoing embodiments, the first sequence extension record portion, the second sequence extension record portion, the third sequence extension record portion and / or the higher order extension record portion can be a sequenceable polymer, such as a polynucleotide or a non-nucleic acid sequenceable polymer.

[1564] In another aspect, disclosed herein is a kit comprising: (a) contacting a polypeptide with a first binding agent capable of binding to one or more N-terminal or C-terminal amino acids of the polypeptide, wherein the first binding agent comprises a first coding portion having identification information about the first binding agent, the polypeptide is associated with a recording portion attached to a support, and the polypeptide and / or the recording portion is immobilized to the support; (b) transferring the information of the first coding portion to a recording polymer to produce a first sequence-extended recording portion; (c) contacting the analyte with a second binding agent capable of binding to a different one or more N-terminal, internal, or C-terminal amino acids of the polypeptide, wherein the second binding agent comprises a second coding portion having identification information about the second binding agent; (d) transferring the information of the second coding portion to the first sequence-extended recording portion to produce a second sequence-extended recording portion; and (e) analyzing the second sequence-extended recording portion. In one embodiment, the one or more N-terminal, internal, or C-terminal amino acids and / or the different one or more N-terminal, internal, or C-terminal amino acids of the polypeptide are modified amino acids, such as modified N-terminal amino acids (NTAA).

[1565] In one aspect, a kit is disclosed herein, comprising: (a) contacting a polypeptide with a first binding agent capable of binding to one or more N-terminal or C-terminal amino acids of the polypeptide, wherein the first binding agent comprises a first coding portion having identification information about the first binding agent, the polypeptide is associated with a recording portion attached to a support, and the polypeptide and / or the recording portion is immobilized to the support; (b) transferring the information of the first coding portion to the recording portion to produce a first sequence-extended recording portion; (c) eliminating one or more N-terminal or C-terminal amino acids of the polypeptide to expose a different one or more N-terminal or C-terminal amino acids of the polypeptide; (d) contacting the analyte with a second binding agent capable of binding to a different one or more N-terminal or C-terminal amino acids of the polypeptide, wherein the second binding agent comprises a second coding portion having identification information about the second binding agent; (e) transferring the information of the second coding portion to the first sequence-extended recording portion to produce a second sequence-extended recording portion; and (f) analyzing the second sequence-extended recording portion. In one embodiment, the one or more N-terminal or C-terminal amino acids and / or the different one or more N-terminal or C-terminal amino acids of the polypeptide are modified amino acids, such as modified N-terminal amino acids (NTAA).

[1566] In any of the foregoing embodiments, the recorded portion of step (a) can be a small molecule, or a subunit, monomer, residue or building block of a sequenceable polymer (eg, a polynucleotide or a non-nucleic acid sequenceable polymer).

[1567] In any of the foregoing embodiments, the recording portion of step (a) can be a sequenceable polymer, e.g., a polynucleotide or a non-nucleic acid sequenceable polymer. In some embodiments, the first or second sequence encoding portion comprises a small molecule and / or subunit, monomer, residue, or building block of a sequenceable polymer (e.g., a polynucleotide or a non-nucleic acid sequenceable polymer).

[1568] In any of the foregoing embodiments, the first or second sequence-encoding tag can be a small molecule, or a subunit, monomer, residue or building block of a sequenceable polymer (eg, a polynucleotide or a non-nucleic acid sequenceable polymer).

[1569] In any of the foregoing embodiments, the first sequence extension recording portion or the second sequence extension recording portion can be a sequenceable polymer, eg, a polynucleotide or a non-nucleic acid sequenceable polymer.

[1570] In any of the foregoing embodiments, the kit can further comprise one or more blocking groups. In one embodiment, the one or more blocking groups are capable of generating a characteristic current blockade signal when the sequenceable polymer passes through the nanopore.

[1571] In any of the foregoing embodiments, the sequenceable polymer may comprise one or more binding groups capable of binding to a binding agent, the binding agent causing the sequenceable polymer to stall as it passes through the nanopore.

[1572] In any of the foregoing embodiments, the sequenceable polymer can comprise one or more structures capable of stalling the sequenceable polymer when passing through the nanopore. In one embodiment, the one or more structures comprise a secondary structure, such as an intrinsic metastable secondary structure.

[1573] In any of the foregoing embodiments, the sequenceable polymer can comprise a polymer constructed using phosphoramidite chemistry, or a peptoid polymer, or a combination thereof.

[1574] In another aspect, disclosed herein is a kit comprising any molecule, molecular complex or conjugate, reagent (e.g., chemical or biological reagent), agent, structure (e.g., support, surface, particle or bead), reaction intermediate, reaction product, binding complex or any other article disclosed and / or used in the method described in any of the above embodiments or any combination thereof.

[1575] Any of the above-described kit components, any molecule, molecular complex or conjugate, reagent (e.g., chemical or biological reagent), agent, structure (e.g., support, surface, particle or bead), reaction intermediate, reaction product, binding complex, or any other article of manufacture disclosed and / or used in the exemplary kits and methods can be provided individually or in any suitable combination to form a kit. The kit can optionally include instructions for use, for example, for highly parallel, high-throughput digital analysis (e.g., macromolecular analysis), particularly polypeptide analysis. BRIEF DESCRIPTION OF THE DRAWINGS

[1576] Non-limiting embodiments of the present disclosure are described by way of example with reference to the accompanying drawings, which are schematic and not intended to be drawn to scale. For illustrative purposes, not every component is labeled in every figure, nor is every component that is not necessary for one of ordinary skill in the art to understand the present invention shown in every embodiment of the present disclosure.

[1577] Figure 1A -B: Attach Figure 1ALegends for the functional elements shown in the figures are shown. Thus, in one embodiment, provided herein are recording tags or extended recording tags comprising one or more universal primer sequences (or one or more pairs of universal primer sequences, e.g., one universal primer in a pair at the 5' end of the recording tag and another universal primer in a pair at the 3' end of the recording tag or extended recording tag), one or more barcode sequences, one or more UMI sequences, one or more spacer sequences, and / or one or more encoder sequences (also referred to as coding sequences, e.g., coding sequences of coding tags) capable of identifying the recording tag or extended recording tag among a plurality of recording tags or extended recording tags. In certain embodiments, the extended recording tag comprises (i) a universal primer sequence, a barcode sequence, a UMI sequence, and a spacer (all derived from the unextended recording tag), (ii) one or more tandemly arranged "cassettes," each containing a coding sequence, a UMI sequence, and a spacer for a binding agent, and each containing sequence information from a coding tag, and (iii) another universal primer sequence, which can be provided by the coding tag of the coding agent in the nth binding cycle, where n is an integer representing the number of binding cycles after which the assay is to be read. In one embodiment, after the universal primer sequence is introduced into the extended recording tag, binding cycles can be continued, the extended recording tag can be further extended, and one or more additional universal primer sequences can be introduced. In this case, any combination of universal primer sequences can be used for amplification and / or sequencing of the extended recording tag. In any of the examples shown in this figure, the encoding tag and / or recording tag (and / or bitag, compartment tag, or partition tag, if applicable), or any portion thereof (e.g., universal primer, spacer, UMI, recording tag barcode, encoder sequence, binding cycle-specific barcode) can include or be replaced with a sequenceable polymer, such as a non-nucleic acid sequenceable polymer.

[1578] Figure 1B A general overview of transducing or converting a protein code into a nucleic acid (e.g., DNA) code is shown, wherein multiple proteins or polypeptides are fragmented into multiple peptides, which are then converted into an extended record tag library to represent the multiple peptides. The extended record tags constitute a DNA encoding library (DEL) representing the peptide sequence. The library can be appropriately modified to be sequenced on any next-generation sequencing (NGS) platform. In any of the examples shown in this figure, the coding tag and / or record tag (and / or bitag, compartment tag, or partition tag, if applicable), or any portion thereof (e.g., universal primer, spacer, UMI, record tag barcode, encoder sequence, combined cycle-specific barcode) can include or be replaced with a sequenceable polymer, such as a non-nucleic acid sequenceable polymer.

[1579] Figure 2A-D illustrates an example of analyzing a polypeptide (e.g., protein) using a multi-cycle binding agent (e.g., antibodies, anticalins, N-recognins proteins (e.g., ClpSs, or UBR box proteins), etc., and variants / homologs thereof, and aptamers, etc.) according to the methods disclosed herein, which comprises a coding tag that interacts with an immobilized protein, and the immobilized protein is co-localized or co-labeled with a single or multiple recording tags. In this example, the recording tag comprises a universal priming site, a barcode (e.g., a partitioned barcode, a compartment barcode, and / or a graded barcode), an optional unique molecular identifier (UMI) sequence, and an optional spacer sequence (Sp) for information transfer between the coding tag and the recording tag (or extended recording tag). The compartment tag (Sp) can be constant between all binding cycles, binding agent-specific, or binding cycle number-specific (e.g., for "timed" binding cycles). In this example, the coding tag comprises a sequence that provides for a binding agent (or a class of binding agents, for example, all specifically binding to a terminal amino acid (such as Figure 3 The encoder sequence, optionally a UMI, and a spacer sequence that hybridizes to a complementary spacer sequence on the recording tag facilitate the transfer of the encoded tag information to the recording tag (e.g., by primer extension, also referred to herein as polymerase extension). Ligation can also be used to transfer sequence information, in which case a spacer sequence can be used, but is not required. In any of the examples shown in this figure, the encoding tag and / or the recording tag (and / or bitag, compartment tag, or partition tag, if applicable), or any portion thereof (e.g., universal primer, spacer, UMI, recording tag barcode, encoder sequence, bound cycle-specific barcode) can include or be replaced with a sequenceable polymer, such as a non-nucleic acid sequenceable polymer.

[1580] Figure 2AThe process of generating an extended record tag by binding a cognate binder to an analyte (e.g., a protein or protein complex) and the transfer of corresponding information from the binder's encoding tag to the analyte's record tag is shown. 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, including the encoder sequence from "n" binding cycles that provides identification information for the binder (e.g., antibody 1 (Ab1), antibody 3 (Ab3), ... antibody "n" (Abn)), the barcode / optional UMI sequence from the record tag, the optional UMI sequence from the binder encoding tag, and flanking universal primer sequences at each end of the library construct to facilitate amplification and analysis by digital next-generation sequencing. In any of the examples shown in this figure, the encoding tag and / or recording tag (and / or bitag, compartment tag, or partition tag, if applicable), or any portion thereof (e.g., universal primer, spacer, UMI, recording tag barcode, encoder sequence, binding cycle-specific barcode) can include or be replaced with a sequenceable polymer, such as a non-nucleic acid sequenceable polymer.

[1581] Figure 2B A schematic example of labeling proteins with DNA barcoded record tags is shown. In the upper figure, N-hydroxysuccinimide (NHS) is an amine-reactive coupling agent, and dibenzocyclooctyl (DBCO) is a strained alkyne used for "click" coupling to the surface of a solid substrate. In this scheme, the record tag is coupled to the epsilon amine of a lysine (K) residue (and optionally the N-terminal amino acid) of a protein via an NHS group. In the bottom figure, the epsilon amine of a lysine (K) residue is labeled with a heterobifunctional linker NHS-alkyne to produce an alkyne "click" group. Azide-labeled DNA record tags can then be easily attached to these reactive alkyne groups via standard click chemistry. In addition, DNA record tags can also be designed with an orthogonal methyltetrazine (mTet) group for downstream coupling to a trans-cyclooctene (TCO)-derived sequencing substrate via an inverse electron demand Diels-Alder (iEDDA) reaction. In any of the examples shown in this figure, the encoding tag and / or recording tag (and / or bitag, compartment tag, or partition tag, if applicable), or any portion thereof (e.g., universal primer, spacer, UMI, recording tag barcode, encoder sequence, binding cycle-specific barcode) can include or be replaced with a sequenceable polymer, such as a non-nucleic acid sequenceable polymer.

[1582] Figure 2CAn example of a protein analysis method using a record tag is shown. In the top figure, the analyte, such as a protein macromolecule, is immobilized on a solid support by a capture agent and optionally by cross-linking. The protein or capture agent can be co-located with the record tag or labeled with the record tag. In the bottom figure, the protein associated with the record tag is directly immobilized on the solid support. In any of the examples shown in the figure, the coding tag and / or the record tag (and / or the dual tag, compartment tag or partition tag, if applicable), or any portion thereof (e.g., a universal primer, a spacer, a UMI, a record tag barcode, an encoder sequence, a binding cycle-specific barcode) can include or be replaced by a sequenceable polymer, such as a non-nucleic acid sequenceable polymer.

[1583] Figure 2D Shown is an overall workflow example of a simple protein immunoassay using DNA encoding of homologous binders and sequencing of extended record tags. Proteins can be barcoded (i.e., indexed) by record tags and pooled before cyclic binding analysis, greatly increasing sample throughput and saving binding reagents. This method is an effective digital, simpler and more scalable method for performing reverse phase protein assay (RPPA), allowing protein levels (e.g., expression levels) in a large number of biological samples to be measured simultaneously in a quantitative manner. In any of the examples shown in the figure, the coding tag and / or record tag (and / or double tag, compartment tag or partition tag, if applicable), or any portion thereof (e.g., universal primer, spacer, UMI, record tag barcode, encoder sequence, combined with cycle-specific barcode) can include or be replaced with a sequenceable polymer, such as a non-nucleic acid sequenceable polymer.

[1584] Figure 3AD shows a process for degradation-based polypeptide sequence determination by constructing an extended record tag (e.g., a DNA sequence) representing the polypeptide sequence. This is achieved by an Edman degradation-like method using a cyclic process (e.g., N-terminal amino acid (NTAA) binding), transferring the encoded tag information to the record tag attached to the polypeptide, cleaving the terminal amino acid (e.g., NTAA cleavage), and repeating the method in a cyclic manner, e.g., all on a solid support. Provided are exemplary structures for constructing extended record tags from the N-terminal degradation of a peptide: (A) the N-terminal amino acid of the peptide is labeled (e.g., with a phenylthiocarbamoyl (PTC), dinitrophenyl (DNP), sulfonylnitrophenyl (SNP), acetyl, or guanidino group); (B) a binding agent is shown with a coding tag associated with the labeled NTAA; (C) a polypeptide is shown that is bound to a solid support (e.g., a bead) and associated with the record tag (e.g., via a trifunctional linker), wherein upon binding of the binding agent to the NTAA of the polypeptide, the information of the coding tag is transferred to the record tag (e.g., by primer extension, including single-stranded ligation, double-stranded ligation, blunt-end ligation, or sticky-end ligation) to produce an extended record tag; (D) the labeled NTAA is cleaved chemically or biologically (e.g., enzymatically) 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 universal priming sites 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 recording tag design, and the reverse universal priming site (e.g., Illumina's P7-S2' sequence) can be added in the final step of the recording tag extension. The final step can be completed independently of the binding agent. In any of the examples shown in this figure, the encoding tag and / or recording tag (and / or bitag, compartment tag, or partition tag, if applicable), or any portion thereof (e.g., universal primer, spacer, UMI, recording tag barcode, encoder sequence, binding cycle-specific barcode) can include or be replaced with a sequenceable polymer, such as a non-nucleic acid sequenceable polymer.

[1585] Figure 4A -B shows an exemplary protein sequencing workflow according to the methods disclosed herein. Figure 4A An exemplary workflow is shown with alternative models outlined in light grey dashed lines, where specific embodiments are shown as boxes linked by arrows. Alternative models for each step of the process are shown in the box below the arrow. Figure 4BThe option of performing cyclic binding and encoding tag information transfer steps to improve information transfer efficiency is shown. Multiple recording tags can be used for each molecule. In addition, for a given binding event, the encoding tag information can be transferred to the recording tag multiple times, or alternatively, a surface amplification step can be used to generate copies of the extended recording tag library, and so on. In any of the examples shown in this figure, the encoding tag and / or recording tag (and / or dual tag, compartment tag or partition tag, if applicable), or any portion thereof (e.g., universal primer, spacer, UMI, recording tag barcode, encoder sequence, binding cycle-specific barcode) can include or be replaced by a sequenceable polymer, such as a non-nucleic acid sequenceable polymer.

[1586] Figure 5 Figures AB show an overview of an exemplary construction of an extended recording tag using primer extension to transfer the identification information of a coding tag of a binding agent to a recording tag associated with an analyte, such as a macromolecule (e.g., a polypeptide), to produce an extended recording tag. The coding tag, comprising a unique coding sequence with identification information about the binding agent, is optionally flanked at each end by a common spacer sequence (Sp'). Figure 5 A shows a NTAA binding agent, which includes a coding tag that is bound to the NTAA of a polypeptide, which is labeled with a record tag and connected to a bead. The record tag is annealed with the coding tag through a complementary spacer sequence (Sp annealing to Sp'), and by using the spacer (Sp) as a primer site, a primer extension reaction mediates the transfer of the information of the coding tag to the record tag. The coding tag is shown as a duplex with a single-stranded spacer (Sp') sequence at the end away from the binding agent. This configuration minimizes the hybridization of the coding tag with the internal site 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 one or more oligonucleotides (complementary to the encoder and spacer sequences) to prevent the hybridization of the coding tag with the internal record tag sequence elements. Figure 5 B shows the final extended recording tag produced after "n" binding cycles ("***" indicates intervening binding cycles not shown in the extended recording tag) and the transfer of the encoding tag information and the addition of a universal priming site at the 3'-end. In any of the examples shown in this figure, the encoding tag and / or recording tag (and / or ditag, compartment tag, or partition tag, if applicable), or any portion thereof (e.g., universal primer, spacer, UMI, recording tag barcode, encoder sequence, binding cycle-specific barcode) can include or be replaced by a sequenceable polymer, such as a non-nucleic acid sequenceable polymer.

[1587] Figure 6The transfer of coding tag information to an extended recording tag by enzyme ligation is shown. Two different analytes and their respective recording tags are shown, wherein the recording tag extension is carried out in parallel. The connection can be facilitated by designing a double-stranded coding tag so that the spacer sequence (Sp') has a "sticky end" protruding on one chain that anneals to the complementary spacer sequence (Sp) on the recording tag. The length of the "sticky end" can be 0-8 bases, for example, about 2-4 bases. The complementary chain of the double-stranded coding tag transfers the information to the recording tag after connection with the recording tag. The complementary chain may contain another spacer sequence, which may be the same as or different from the Sp of the recording tag before connection. When connection is used to extend the recording tag, the direction of extension can be 5' to 3' as shown in the figure, or optionally 3' to 5'. In any of the examples shown in this figure, the encoding tag and / or recording tag (and / or bitag, compartment tag, or partition tag, if applicable), or any portion thereof (e.g., universal primer, spacer, UMI, recording tag barcode, encoder sequence, binding cycle-specific barcode) can include or be replaced with a sequenceable polymer, such as a non-nucleic acid sequenceable polymer.

[1588] 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 extended recording tag produced 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. In any of the examples shown in this figure, the encoding tag and / or recording tag (and / or bitag, compartment tag, or partition tag, if applicable), or any portion thereof (e.g., universal primer, spacer, UMI, recording tag barcode, encoder sequence, binding cycle-specific barcode) can include or be replaced with a sequenceable polymer, such as a non-nucleic acid sequenceable polymer.

[1589] Figure 8 AB shows an exemplary method for writing post-translational modifications (PTMs) of a polypeptide into an extended record tag before degradation of the N-terminal amino acid. Figure 8A: A binding agent containing a coding tag that contains identification information for the binding agent (e.g., a phosphotyrosine antibody containing a coding tag that contains identification information for the phosphotyrosine antibody) can bind to a polypeptide. If phosphotyrosine is present in the polypeptide labeled with the recording tag, as shown in the figure, after the phosphotyrosine antibody binds to the phosphotyrosine, the coding tag and the recording tag anneal via complementary spacer sequences, and the coding tag information is transferred to the recording tag, generating an extended recording tag. Figure 8 B: Extended record labels can contain the primary amino acid sequence of the peptide (e.g., "aa l ", "aa2", "aa3"..., "aa N In any of the examples shown in the figure, the encoding tag and / or recording tag (and / or bitag, compartment tag, or partition tag, if applicable), or any portion thereof (e.g., universal primer, spacer, UMI, recording tag barcode, encoder sequence, binding cycle-specific barcode) can include or be replaced by a sequenceable polymer, such as a non-nucleic acid sequenceable polymer.

[1590] Figure 9 Figures AB show a process of multiple cycles in which a binding agent binds to an analyte (e.g., a macromolecule such as a polypeptide) and transfers information from a coded tag attached to the binding agent to a single recording tag among a plurality of recording tags (e.g., co-localized on a single analyte attached to a site on a solid support (e.g., a bead), thereby generating a plurality of extended recording tags that collectively represent analyte information (e.g., presence or absence in a sample, level or amount, binding to a library of binding agents, activity or reactivity, amino acid sequence, post-translational modification, sample origin, or any combination thereof). In this figure, for exemplary purposes only, the analyte is a polypeptide, and each cycle involves binding a binding agent to the N-terminal amino acid (NTAA), recording the binding event by transferring the coded tag information to the recording tag, and then removing the NTAA to expose a new NTAA. Figure 9A shows a plurality of recording tags (e.g., comprising a universal forward priming sequence and a UMI) on a solid support, which can be used for binding agents that bind to an analyte. A single recording tag has a common spacer sequence (Sp) that is complementary to a common spacer sequence within the coding tag of the binding agent, which can be used to initiate an extension reaction to transfer the coded tag information to the recording tag. For example, multiple recording tags can be co-localized with the analyte on the support, and some recording tags can be closer to the analyte than ...

Claims

1. A method for analyzing a plurality of polypeptides in a sample, comprising: (a) linking each polypeptide of the plurality of polypeptides to a nucleic acid tag to generate a nucleic acid-polypeptide chimera; (b) hybridizing the nucleic acid tag in the nucleic acid-polypeptide chimera to a capture nucleic acid attached to a solid support; (c) covalently immobilizing the nucleic acid-polypeptide chimera to the solid support; and (d) contacting the covalently linked nucleic acid-polypeptide chimera with a plurality of binding agents, wherein each of the plurality of binding agents binds to a component of one or more polypeptides in the plurality of polypeptides, and each of the plurality of binding agents comprises: (i) a nucleic acid-encoded tag having identification information about the binding agent, or (ii) a detectable tag; thereby analyzing the one or more polypeptides in the covalently linked nucleic acid-polypeptide chimera. 2 . The method of claim 1 , wherein each polypeptide of the plurality of polypeptides is linked to the nucleic acid tag via an N-terminal amino acid (NTAA) residue or a C-terminal amino acid (NTAA) residue. 3 . The method of claim 1 , wherein each polypeptide of the plurality of polypeptides is linked to the nucleic acid tag via a lysine residue.

4. The method of claim 1, wherein the plurality of binding agents comprises three different binding agents, each binding agent being selective for a different component of one or more polypeptides in the plurality of polypeptides.

5. The method of claim 4, wherein each of the at least three different binding agents is configured to bind to an NTAA residue or a CTAA residue of one or more polypeptides of the plurality of polypeptides.

6. The method of claim 1, wherein each of the plurality of binding agents comprises a detectable label comprising a fluorescent group, a color-coded nanoparticle, a quantum dot, or any combination thereof.

7. The method of claim 1, wherein each of the plurality of binding agents comprises a nucleic acid-encoded tag having identification information about the binding agent.

8. The method of claim 1, wherein each polypeptide of the plurality of polypeptides is chemically conjugated to an internal nucleotide of the nucleic acid tag.

9. The method of claim 1, wherein the capture nucleic acid comprises a hairpin region.

10. The method of claim 1, wherein at least some of the plurality of polypeptides are each linked to a plurality of nucleic acid tags, and each polypeptide is fragmented or digested into smaller polypeptides, each of which is linked to an average of one nucleic acid tag.

11. The method of claim 1, wherein the plurality of polypeptides is a proteome or a subset thereof.

12. The method of claim 1, wherein the nucleic acid tag comprises a barcode or a unique molecular identifier (UMI), wherein the barcode comprises an analyte barcode, a compartment barcode, a partition barcode, a sample barcode, a fractionator barcode, or any combination thereof.

13. The method of claim 1, wherein the solid support is a bead or a flow cell.

14. The method of claim 1, wherein the nucleic acid-polypeptide chimeras are randomly attached to the solid support.

15. The method of claim 1, wherein the plurality of polypeptides comprises at least 1,000,000 polypeptides.

16. A kit for analyzing one or more polypeptides from different samples, comprising: (a) one or more nucleic acid record tags, which are configured to be directly or indirectly associated with the one or more polypeptides, or directly or indirectly associated with one or more components obtained by cleaving the one or more polypeptides, wherein each of the one or more nucleic acid record tags comprises a unique molecular identifier (UMI) and further comprises a sample barcode, and the sample barcode is configured to distinguish polypeptides from different samples; as well as (b) one or more binding agents, each binding agent comprising: (i) a binding moiety capable of binding to the one or more polypeptides or one or more components thereof; and (ii) a nucleic acid encoding tag attached to the binding moiety, the nucleic acid encoding tag comprising a barcode sequence, the barcode sequence comprising identification information about the binding moiety; The one or more nucleic acid recording tags and the one or more nucleic acid encoding tags are configured to allow the barcode sequence and / or the UMI sequence information to be transferred by ligation or primer extension when each of the binding agents binds to the one or more polypeptides or components thereof, thereby generating one or more extended nucleic acid constructs, each of which comprises a barcode sequence or its complement, a sample barcode or its complement, and a UMI or its complement.

17. A kit for analyzing a polypeptide, comprising: (a) a functionalizing agent capable of modifying the N-terminal amino acid (NTAA) of the polypeptide to generate a functionalized NTAA of the polypeptide; (b) one or more nucleic acid record tags configured to be directly or indirectly associated with the polypeptide, or directly or indirectly associated with a component of the polypeptide obtained by cleaving a functionalized NTAA of the polypeptide, wherein each of the one or more nucleic acid record tags comprises a unique molecular identifier (UMI); and (c) one or more binding agents, each binding agent comprising: (i) a binding moiety capable of binding to the functionalized NTAA of the polypeptide or to a component of the polypeptide obtained by cleaving the functionalized NTAA of the polypeptide; and (ii) a nucleic acid encoding tag attached to the binding moiety, the nucleic acid encoding tag comprising a barcode sequence, the barcode sequence comprising identification information about the binding moiety; Wherein the one or more nucleic acid recording tags and the one or more nucleic acid encoding tags are configured to allow the sequence information of the barcode sequence and / or the UMI to be transferred by ligation or primer extension when each of the binding agents binds to the functionalized NTAA of the polypeptide or to the component of the polypeptide obtained by cleaving the functionalized NTAA of the polypeptide, thereby generating one or more extended nucleic acid constructs, each of which comprises a barcode sequence or its complement, and a UMI or its complement.

18. The kit according to claim 17, wherein the kit further comprises an elimination reagent for removing the functionalized NTAA of the polypeptide.

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