Molecules and methods for iterative polypeptide analysis and processing

Patent metadata

Field	Value
Publication number	US-10852305-B2
Application number	US-202016907813-A
Country	US
Kind code	B2
Filing date	Jun 22, 2020
Priority date	Mar 15, 2013
Publication date	Dec 1, 2020
Grant date	Dec 1, 2020

Abstract

Reagents and methods for the digital analysis of proteins or peptides are provided. Specifically provided herein are proteins for identifying the N-terminal amino acid or N-terminal phosphorylated amino acid of a polypeptide. Also, an enzyme for use in the cleavage step of the Edman degradation reaction and a method for using this enzyme are described.

First claim

What is claimed is:

1. A method for identifying a N-terminal amino acid of a polypeptide, the method comprising:
(a) contacting the polypeptide with one or more fluorescently labeled N-terminal amino acid binding proteins (NAABs);
(b) detecting fluorescence of the NAAB that is bound to the N-terminal amino acid of the polypeptide;
(c) identifying the N-terminal amino acid of the polypeptide based on the detected fluorescence; and
(d) cleaving the identified N-terminal amino acid of the polypeptide.

2. The method of claim 1 , wherein the cleaving of step (d) is performed using a protease.

3. The method of claim 2 , wherein the protease comprises an isolated, synthetic, or recombinant Edman degradation enzyme.

4. The method of claim 1 , wherein the cleavage in step (d) catalyzes the cleavage in an aqueous buffer and at a neutral pH.

5. The method of claim 1 , wherein the cleavage in step (d) does not employ an acidic condition and elevated temperature.

6. The method of claim 2 , wherein the protease allows for promiscuous cleavage of diverse N-terminal amino acids.

7. The method of claim 3 , wherein the Edman degradation enzyme cleaves the N-terminal amino acid by nucleophilic attack of the thiourea sulfur atom on the carbonyl group of the scissile peptide bond.

8. The method of claim 3 , wherein the Edman degradation enzyme is configured for substrate assisted catalysis.

9. The method of claim 1 , wherein the cleavage in step (d) comprises treating the polypeptide with two or more enzymes.

10. The method of claim 9 , wherein the two or more enzymes comprise an enzyme for removal of proline from a polypeptide.

11. The method of claim 1 , wherein step (d) cleaves a modified N-terminal amino acid from the polypeptide.

12. The method of claim 1 , wherein the NAAB binds to an N-terminal amino acid with a post translational-modification.

13. The method of claim 12 , wherein N-terminal amino acid with the post translational-modification comprises a phosphorylated N-terminal amino acid.

14. The method of claim 1 , wherein a single NAAB is configured to identify multiple amino acids.

15. The method of claim 1 , which comprises simultaneously sequencing a plurality of polypeptides.

16. The method of claim 1 , which further comprises immobilizing the polypeptide or polypeptides on a substrate prior to the contacting in step (a).

17. The method of claim 1 , wherein the contacting in step (a) comprises contacting the polypeptide with a single type of NAAB that selectively binds to a single type of N-terminal amino acid residue.

18. The method of claim 1 , wherein the contacting in step (a) comprises contacting the polypeptide with a first type of NAAB and a second type of NAAB, wherein the first type of NAAB selectively binds to a first type of N-terminal amino acid residue and the second type of NAAB selectively binds to a second type of N-terminal amino acid residue different from the first type of N-terminal amino acid residue.

19. The method of claim 18 , wherein the first type of NAAB is coupled to a first fluorophore and the second type of NAAB is coupled to a second fluorophore, wherein the first and second fluorophores have different fluorescence emission spectra.

20. The method of claim 1 , wherein a plurality of NAABs are introduced in a step-wise fashion.

21. The method of claim 1 , wherein the polypeptide is contacted with a mixture of two or more types of NAABs that each selectively binds to different amino acid residues.

22. The method of claim 1 , further comprising removing the NAAB from the polypeptide by contacting the polypeptide with a wash buffer that causes dissociation of the NAAB bound to the N-terminal amino acid of the polypeptide.

23. The method of claim 1 , wherein steps (a) through (d) are repeated until the polypeptide or a fragment thereof is sequenced.

Assignees

• Univ Washington

Inventors

• Havranek James J
• Borgo Benjamin

Classifications

• C12N9/52

  derived from bacteria {or Archaea} · CPC title

• C12N9/641

  Cysteine endopeptidases (3.4.22) · CPC title

• G01N33/6824 Primary

  involving N-terminal degradation, e.g. Edman degradation · CPC title

• C12Y601/01021

  Histidine-tRNA ligase (6.1.1.21) · CPC title

• C12N9/485

  Exopeptidases (3.4.11-3.4.19) · CPC title