NZ787458A - Macromolecule analysis employing nucleic acid encoding - Google Patents
Macromolecule analysis employing nucleic acid encodingInfo
- Publication number
- NZ787458A NZ787458A NZ787458A NZ78745817A NZ787458A NZ 787458 A NZ787458 A NZ 787458A NZ 787458 A NZ787458 A NZ 787458A NZ 78745817 A NZ78745817 A NZ 78745817A NZ 787458 A NZ787458 A NZ 787458A
- Authority
- NZ
- New Zealand
- Prior art keywords
- nucleic acid
- binding agent
- peptides
- tags
- peptide
- Prior art date
Links
- 108020004707 nucleic acids Proteins 0.000 title claims abstract description 36
- 102000039446 nucleic acids Human genes 0.000 title claims abstract description 36
- 150000007523 nucleic acids Chemical class 0.000 title claims abstract description 36
- 229920002521 macromolecule Polymers 0.000 title abstract description 4
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 56
- 102000004196 processed proteins & peptides Human genes 0.000 claims abstract description 37
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 16
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 16
- 229920001184 polypeptide Polymers 0.000 claims abstract description 14
- 238000000034 method Methods 0.000 claims abstract 21
- 239000011230 binding agent Substances 0.000 claims 31
- 125000001429 N-terminal alpha-amino-acid group Chemical group 0.000 claims 19
- 239000007787 solid Substances 0.000 claims 8
- 239000011324 bead Substances 0.000 claims 4
- 125000000539 amino acid group Chemical group 0.000 claims 3
- 150000001413 amino acids Chemical class 0.000 claims 3
- 238000000137 annealing Methods 0.000 claims 2
- 239000013043 chemical agent Substances 0.000 claims 2
- 230000003100 immobilizing effect Effects 0.000 claims 2
- 238000003203 nucleic acid sequencing method Methods 0.000 claims 2
- 238000000638 solvent extraction Methods 0.000 claims 2
- 102000004400 Aminopeptidases Human genes 0.000 claims 1
- 108090000915 Aminopeptidases Proteins 0.000 claims 1
- 108091023037 Aptamer Proteins 0.000 claims 1
- 125000003275 alpha amino acid group Chemical group 0.000 claims 1
- 239000003153 chemical reaction reagent Substances 0.000 claims 1
- 239000002773 nucleotide Substances 0.000 claims 1
- 125000003729 nucleotide group Chemical group 0.000 claims 1
- 239000000126 substance Substances 0.000 claims 1
- 230000004481 post-translational protein modification Effects 0.000 description 2
- 230000008827 biological function Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 230000004044 response Effects 0.000 description 1
Abstract
method for analyzing macromolecules, including peptides, polypeptides, and proteins, employing nucleic acid encoding is disclosed.
Description
MACROMOLECULE IS EMPLOYING NUCLEIC ACID ENCODING This application is a divisional of New Zealand patent application 747941, which is the National Phase Entry in New Zealand of pct international application (published as are incorporated herein by reference in their entireties.
STATEMENT REGARDING SEQUENCE LISTING The Sequence Listing associated with this application is provided in text format in lieu of a paper copy, and is hereby incorporated by reference into the specification. The name of the text file containing the Sequence g is _401WO_SEQUENCE_LISTING.txt. The text file is 38.7 KB, was created on May 2, 2017, and is being submitted electronically via b.
BACKGROUND Technical Field This disclosure generally relates to analysis of macromolecules, including es, polypeptides, and proteins, employing barcoding and nucleic acid ng of molecular recognition events.
Description of the Related Art Proteins play an integral role in cell biology and physiology, performing and facilitating many different biological functions. The repertoire of different protein molecules is extensive, much more complex than the transcriptome, due to additional diversity introduced by post-translational modifications (PTMs). Additionally, proteins within a cell cally change (in expression level and modification state) in response to the environment, physiological state, and disease state. Thus, ns contain a vast amount of nt information that is largely unexplored, especially
Claims (18)
1. A method for analyzing a peptide comprising more than two amino acid residues, the method comprising the steps of: (a) providing the peptide comprising more than two amino acid residues and an associated nucleic acid recording tag, both joined to a solid support; (b) contacting the peptide with a first binding agent capable of binding to the peptide, wherein the first binding agent comprises a first nucleic acid coding tag that comprises identifying information regarding the first binding agent; (c) following binding of the first binding agent to the peptide, transferring the identifying information regarding the first binding agent from the first nucleic acid coding tag to the nucleic acid recording tag to generate an extended nucleic acid recording tag joined to the solid support, wherein the transferring comprises primer extension or ligation; and (d) analyzing the extended nucleic acid recording tag by a nucleic acid sequencing method and obtaining the identifying information regarding the first binding agent to provide information regarding the peptide, thereby analyzing the peptide.
2. The method of claim 1, wherein step (a) comprises providing a plurality of peptides and associated nucleic acid recording tags joined to the solid support; and before analyzing the plurality of peptides by the steps (a)-(d), the plurality of peptides are associated with nucleic acid recording tags by the steps of: (q) partitioning a plurality of proteins or polypeptides from a sample into a plurality of compartments, wherein each compartment comprises a plurality of compartment tags optionally attached to beads, wherein each nucleic acid compartment tag of the plurality of compartment tags is the same within an individual compartment and different from the compartment tags of other compartments; (r) contacting the partitioned proteins or polypeptides with pluralities of compartment tags within the plurality of compartments under conditions sufficient to permit annealing or joining of the partitioned proteins or polypeptides to nucleic acid compartment tags, thereby generating a plurality of recording tag-labeled proteins or polypeptides; (s) fragmenting the plurality of proteins or polypeptides to obtain peptides, wherein the fragmenting is performed before or after step (r); (t) collecting the recording tag-labeled peptides from the plurality of compartments; and (u) immobilizing the recording tag-labeled peptides on the solid support.
3. The method of claim 2, further comprising releasing compartment tags from the plurality of compartment tags from the beads within the plurality of compartments after step (q) and before step (r).
4. The method of claim 1, wherein step (a) comprises providing a plurality of peptides and associated nucleic acid recording tags joined to the solid support; and before analyzing the plurality of peptides by the steps (a)-(d), the plurality of peptides are associated with nucleic acid recording tags by the steps of: (q) partitioning a plurality of proteins or polypeptides from a sample into a plurality of compartments, wherein each compartment comprises a plurality of compartment tags attached to beads, wherein each compartment tag of the plurality of compartment tags is the same within an individual compartment and different from the compartment tags of other compartments; (r) contacting the partitioned proteins or polypeptides with pluralities of compartment tags within the plurality of compartments under conditions sufficient to permit annealing or joining of the partitioned proteins or polypeptides to compartment tags, thereby generating a plurality of recording tag-labeled proteins or polypeptides; (s) optionally releasing compartment tags from the plurality of compartment tags from the beads; (t) collecting the recording tag-labeled proteins or polypeptides from the plurality of compartments; (u) fragmenting the recording tag-labeled polypeptides to obtain the recording tag-labeled peptides; and (v) immobilizing the recording tag-labeled peptides on the solid support.
5. The method of claim 1, further comprising attaching the peptide to the nucleic acid recording tag joined to the solid support before step (a).
6. The method of claim 1, wherein the first binding agent binds specifically to a post-translationally modified amino acid of the peptide.
7. The method of claim 6, further comprising contacting an amino acid of the peptide with a modifying reagent before step (b) to obtain the post-translationally modified amino acid.
8. The method of claim 1, wherein the identifying information regarding the first binding agent obtained at step (d) is used to determine all or a portion of an amino acid sequence of the peptide.
9. The method of claim 1, wherein the nucleic acid recording tag is joined to the peptide via a non-nucleotide chemical moiety.
10. The method of claim 1, wherein the first binding agent comprises a polypeptide, a protein or an aptamer.
11. The method of claim 1, wherein the first binding agent is capable of binding to the N-terminal amino acid (NTAA) of the peptide.
12. The method of claim 1, further comprising: (ab) modifying the N- terminal amino acid (NTAA) of the peptide with a chemical agent to yield a modified NTAA, wherein the first binding agent is capable of binding to the modified NTAA, and the step (ab) is performed before step (b) and after step (a).
13. The method of claim 12, further comprising: (cd) removing the modified NTAA to generate a new NTAA of the peptide, wherein the step (cd) is performed before step (d) and after step (c).
14. The method of claim 13, wherein the step (cd) is performed by a modified aminopeptidase.
15. The method of claim 13, further comprising repeating steps (ab), (b) and (c) sequentially after the step (cd) by generating a newly modified NTAA from the new NTAA; by replacing the first binding agent with a second binding agent capable of binding to the newly modified NTAA, wherein the second binding agent comprises a second nucleic acid coding tag that comprises identifying information regarding the second binding agent; and by transferring, following binding of the second binding agent to the newly modified NTAA, the identifying information regarding the second binding agent from the second nucleic acid coding tag to the extended nucleic acid recording tag generating a second order extended nucleic acid recording tag, wherein the transferring comprises primer extension or ligation; and wherein the second order extended nucleic acid recording tag is analyzed and the identifying information regarding the second binding agent is obtained in step (d).
16. The method of claim 1, further comprising: after steps (a)-(c) and before step (d) performing sequentially the following steps: (i) modifying an N-terminal amino acid (NTAA) of the peptide with a chemical agent to yield a modified NTAA; (ii) contacting the peptide with a second binding agent capable of binding to the modified NTAA, wherein the second binding agent comprises a second nucleic acid coding tag that comprises identifying information regarding the second binding agent; (iii) following binding of the second binding agent to the modified NTAA, transferring the identifying information regarding the second binding agent from the second nucleic acid coding tag to the extended nucleic acid recording tag to generate a further extended nucleic acid recording tag, wherein the transferring comprises primer extension or ligation; wherein the further extended nucleic acid recording tag is analyzed and the identifying information regarding the second binding agent is obtained in step (d).
17. The method of claim 16, further comprising removing the modified NTAA to expose a new NTAA.
18. A method for analyzing a plurality of peptides each comprising more than two amino acid residues, the method comprising the steps of: (a) providing a plurality of peptides and associated nucleic acid recording tags, both joined to a solid support, wherein each nucleic acid recording tag comprises a unique molecular identifier (UMI) that identifies an associated peptide of the plurality of peptides; (b) contacting the plurality of peptides with a plurality of binding agents capable of binding to the plurality of peptides, wherein each binding agent comprises a nucleic acid coding tag that comprises identifying information regarding the binding agent; (c) following binding of binding agents from the plurality of binding agents to peptides from the plurality of peptides, transferring the identifying information of the peptide associated nucleic acid recording tags to the nucleic acid coding tags of the binding agents that are bound to the peptides to generate extended nucleic acid coding tags, wherein the transferring comprises primer extension or ligation; (d) collecting the extended nucleic acid coding tags; (e) optionally repeating steps (b) – (d) for one or more binding cycles; and (f) analyzing the collection of extended nucleic acid coding tags, wherein analyzing comprises a nucleic acid sequencing method, and obtaining the identifying information regarding the binding agents together with UMIs that identify the associated peptides to provide information regarding the plurality of peptides, thereby analyzing the plurality of peptides.
Publications (1)
| Publication Number | Publication Date |
|---|---|
| NZ787458A true NZ787458A (en) | 2022-04-29 |
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