Identifying peptides at the single molecule level

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 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 acid that is not lysine;

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 amino acid that is not lysine is identified.

40. The method of claim 39 , 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.

41. The method of claim 39 , wherein the N-terminal amino acids are removed in step b) by an Edman degradation reaction.

42. The method of claim 39 , wherein the peptides are immobilized via cysteine residues.

43. A method of generating and treating peptides, comprising:

a) digesting a protein preparation with an agent that cleaves after a specific amino acid residue so as to generate a plurality of peptides, each peptide comprises an N-terminal amino acid and internal amino acids, at least a portion of said internal amino acids of said peptides comprising lysine, at least a portion of said peptides comprising said specific amino acid residue at the C-terminus;

b) labeling said plurality of peptides such that each lysine is labeled with a label, said label producing a signal for each peptide;

c) immobilizing said labeled peptides on a solid support;

d) treating said plurality of immobilized peptides under conditions such that each N-terminal amino acid of each peptide is removed; and

e) detecting the signal for each peptide at the single molecule level.

44. The method of claim 43 , wherein said agent is an enzyme.

45. The method of claim 44 , wherein said enzyme is a specific endopeptidase.

46. The method of claim 45 , wherein said endopeptidase is peptidase GluC.

47. The method of claim 43 , wherein said agent is a chemical agent.

48. The method of claim 47 , wherein said chemical agent is cyanogen bromide.

49. The method of claim 43 , wherein said label is a fluorescent label.

50. The method of claim 43 , wherein the removal in step d) said N-terminal amino acid of each peptide reacted with a phenyl isothiocyanate derivative.

51. The method of claim 43 , wherein the removal of said N-terminal amino acid in step d) is done under conditions such that the remaining peptides each have a new N-terminal amino acid.

52. The method of claim 51 , further comprising the step f) removing the next N-terminal amino acid done under conditions such that the remaining peptides each have a new N-terminal amino acid.

53. The method of claim 52 , further comprising the step g) detecting the next signal for each peptide at the single molecule level.

54. The method of claim 53 , wherein the N-terminal amino acid removing step and the detecting step are successively repeated from 1 to 20 times.

55. The method of claim 54 , wherein the repetitive detection of signal for each peptide at the single molecule level results in a pattern.

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

57. The method of claim 9 , wherein the single-peptide pattern is compared to the proteome of an organism, using said specific amino acid residue at the C-terminus, to identify the peptide.