Identifying peptides at the single molecule level

Patent metadata

Field	Value
Publication number	US-9625469-B2
Application number	US-201214128247-A
Country	US
Kind code	B2
Filing date	Jun 22, 2012
Priority date	Jun 23, 2011
Publication date	Apr 18, 2017
Grant date	Apr 18, 2017

Abstract

The present invention relates to methods for identifying amino acids in peptides. In one embodiment, the present invention contemplates labeling the N-terminal amino acid with a first label and labeling an internal amino acid with a second label. In some embodiments, the labels are fluorescent labels. In other embodiments, the internal amino acid is lysine. In other embodiments, amino acids in peptides are identified based on the fluorescent signature for each peptide at the single molecule level.

First claim

We claim:

1. A method of treating peptides, comprising:
a) providing a plurality of peptides immobilized on a solid support, each peptide comprising an N-terminal amino acid and internal amino acids, said internal amino acids comprising lysine, each lysine labeled with a label, and said label producing a signal for each peptide;
b) treating said plurality of immobilized peptides under conditions such that each N-terminal amino acid of each peptide is removed; and
c) detecting the signal for each peptide at the single molecule level.

2. The method of claim 1 , wherein said label is a fluorescent label.

3. The method of claim 1 , wherein the removal in step b) said N-terminal amino acid of each peptide reacted with a phenyl isothiocyanate derivative.

4. The method of claim 1 , wherein the removal of said N-terminal amino acid in step b) is done under conditions such that the remaining peptides each have a new N-terminal amino acid.

5. The method of claim 4 , further comprising the step d) removing the next N-terminal amino acid done under conditions such that the remaining peptides each have a new N-terminal amino acid.

6. The method of claim 5 , further comprising the step e) detecting the next signal for each peptide at the single molecule level.

7. The method of claim 6 , wherein the N-terminal amino acid removing step and the detecting step are successively repeated from 1 to 20 times.

8. The method of claim 7 , wherein the repetitive detection of signal for each peptide at the single molecule level results in a pattern.

9. The method of claim 8 , wherein the pattern is unique to a single-peptide within the plurality of immobilized peptides.

10. The method of claim 9 , wherein the single-peptide pattern is compared to the proteome of an organism to identify the peptide.

11. The method of claim 6 , wherein the intensity of said labels are measured amongst said plurality of immobilized peptides.

12. The method of claim 1 , wherein the N-terminal amino acids are removed in step b) by an Edman degradation reaction.

13. The method of claim 1 , wherein the peptides are immobilized via cysteine residues.

14. The method of claim 1 , wherein the detecting in step c) is done with optics capable of single-molecule resolution.

15. The method of claim 7 , wherein the degradation step in which removal of the N-terminal amino acid coincides with removal of the label is identified.

16. The method of claim 15 , wherein said removal of the amino acid is measured in step b) is measured as a reduced fluorescence intensity.

17. A method of treating peptides, comprising:
a) providing a plurality of peptides immobilized on a solid support, each peptide comprising an N-terminal amino acid and internal amino acids, said internal amino acids comprising lysine, each lysine labeled with a first label, said first label producing a first signal for each peptide, and said N-terminal amino acid of each peptide labeled with a second label, said second label being different from said first label;
b) treating said plurality of immobilized peptides under conditions such that each N-terminal amino acid of each peptide is removed; and
c) detecting the first signal for each peptide at the single molecule level.

18. The method of claim 17 , wherein said second label is attached via an amine-reactive dye.

19. The method of claim 18 , wherein said second label is selected from the group consisting of fluorescein isothiocyanate, rhodamine isothiocyanate or other synthesized fluorescent isothiocyanate derivative.

20. The method of claim 17 , wherein portions of the emission spectrum of said first label do not overlap with the emission spectrum of said second label.

21. The method of claim 17 , wherein the removal of said N-terminal amino acid in step b) is done under conditions such that the remaining peptides each have a new N-terminal amino acid.

22. The method of claim 21 , further comprising the step d) adding said second label to said new N-terminal amino acids of the remaining peptides.

23. The method of claim 22 , wherein among the remaining peptides the new end terminal amino acid is lysine.

24. The method of claim 22 , further comprising the step e) detecting the next signal for each peptide at the single molecule level.

25. The method of claim 24 , wherein the N-terminal amino acid removing step, the detecting step, and the label adding step to a new N-terminal amino acid are successively repeated from 1 to 20 times.

26. The method of claim 25 , wherein the repetitive detection of signal for each peptide at the single molecule level results in a pattern.

27. The method of claim 26 , wherein the pattern is unique to a single-peptide within the plurality of immobilized peptides.

28. The method of claim 27 , wherein the single-peptide pattern is compared to the proteome of an organism to identify the peptide.

29. The method of claim 24 , wherein the intensity of said first and second labels are measured amongst said plurality of immobilized peptides.

30. The method of claim 17 , wherein the N-terminal amino acids are removed in step b) by an Edman degradation reaction.

31. The method of claim 17 , wherein the peptides are immobilized via cysteine residues.

32. The method of claim 17 , wherein the detecting in step c) is done with optics capable of single-molecule resolution.

33. The method of claim 23 , wherein the degradation step in which removal of second label coincides with removal of first label is identified.

34. The method of claim 33 , wherein said removal of the amino acid is measured in step b is measured as a reduced fluorescence intensity.

35. A method of identifying amino acids in peptides, comprising:
a) providing a plurality of peptides immobilized on a solid support, each peptide comprising an N-terminal amino acid and internal amino acids, said internal amino acids comprising lysine, each lysine labeled with a first label, said first label producing a first signal for each peptide, and said N-terminal amino acid of each peptide labeled with a second label, said second label being different from said first label, wherein a subset of said plurality of peptides comprise an N-terminal lysine having both said first and second label;
b) treating said plurality of immobilized peptides under conditions such that each N-terminal amino acid of each peptide is removed; and
c) detecting the first signal for each peptide at the single molecule level under conditions such that said subset of peptides comprising an N-terminal lysine having both said first and second label is identified.

36. The method of claim 35 , wherein the removal of said N-terminal amino acid in step b) is done under conditions such that the remaining peptides each have a new N-terminal amino acid.

37. The method of claim 35 , wherein the N-terminal amino acids are removed in step b) by an Edman degradation reaction.

38. The method of claim 35 , wherein the peptides are immobilized via cysteine residues.

39. A method of identifying amino acids in peptides, comprising:
a) providing a plurality of peptides immobilized on a solid support, each peptide comprising an N-terminal amino acid and inter

Assignees

• Marcotte Edward
• Swaminathan Jagannath
• Ellington Andrew
• Anslyn Eric
• Univ Texas

Inventors

• Marcotte Edward
• Swaminathan Jagannath
• Ellington Andrew
• Anslyn Eric

Classifications

• G01N2570/00

  Omics, e.g. proteomics, glycomics or lipidomics; Methods of analysis focusing on the entire complement of classes of biological molecules or subsets thereof, i.e. focusing on proteomes, glycomes or lipidomes · CPC title

• G01N33/6824 Primary

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