Triazine derivatives as universal peptide isotope tag reagents (U-PIT)

• US 20040115821A1
• Filed: 12/13/2002
• Published: 06/17/2004
• Est. Priority Date: 12/13/2002
• Status: Active Grant

• Current Assignee: Agilent Technologies, Inc.
• Original Assignee: Agilent Technologies, Inc.
• Inventors: Robotti, Karla M., Apffel, James Alexander Jr.
• Granted Patent: US 7,425,451 B2
• Time in Patent Office: Days
• US Class Current: 436/86
• CPC Class Codes: C07D 401/14 containing three or more hetero rings
• CPC Class Codes: C07D 403/12 linked by a chain containing hetero atoms as chain links

Abstract

Compounds, compositions, methods for sequencing proteins and peptides, and methods for identifying proteins and peptides in a mixture, are disclosed. Compounds of formula A-B-C wherein A is a nucleophilic reactive group, B is a detectable moiety capable of being isotopically labeled, and C is a charge replacement group, are used to label the peptides at the N-terminus or the C-terminus. The tagged peptides can then be analyzed by mass spectroscopy.

Claims

1. A compound of formula:

A-B-C

wherein;

A is a nucleophilic reactive group;

B is a detectable moiety capable of being isotopically labeled; and

C is a charge replacement group.

2. The compound of claim 1, wherein A comprises:

3. The compound of claim 2, wherein G is succinimide.

4. The compound of claim 2, wherein G is glutarimide.

5. The compound of claim 2, wherein X₁ and X₃ are O.

6. The compound of claim 5, wherein X₂ is O.

7. The compound of claim 5, wherein X₂ is a direct bond.

8. The compound of claim 1, wherein A is:

9. The compound of claim 1, wherein A is:

10. The compound of claim 1, wherein A is:

11. The compound of claim 1, wherein A is:

12. The compound of claim 1, wherein A is:

13. The compound of claim 1, wherein B is selected from the group consisting of lowerl alkyl, ether, aryl, and heteroaryl.

14. The compound of claim 13, wherein B is ether.

15. The compound of claim 14, wherein ether is polyethyleneglycol.

16. The compound of claim 13, wherein B is heteroaryl.

17. The compound of claim 16, wherein the heteroaryl is selected from the group consisting of substituted or unsubstitued 1,3,5-triazine, 1,2,5-triazine, 1,2,3-triazine, pyridine, imidazole, and thiophene.

18. The compound of claim 1, wherein B comprises the formula:

19. The compound of claim 18, wherein X and X′

are NH, R is CH₂CH₂, and R′

is ethyl.

20. The compound of claim 18, wherein X and X′

are NH, R is CH₂CH₂OCH₂CH₂, and R′

is ethyl.

21. The compound of claim 18, wherein at least one atom in B is replaced with its isotope.

22. The compound of claim 21, wherein the isotope is selected from the group consisting of ²H ³H, ¹³C, ¹⁴C, ³²P, ³⁴S, ³⁵S, ³⁶Cl, ³⁷Cl, ¹⁸O, ¹⁵N, ⁸¹Br, ¹²³I, ¹²⁵I and ¹³¹I.

23. The compound of claim 18, wherein all atoms in B are replaced with their isotopes.

24. The compound of claim 18, wherein mass difference between the isotopically labeled and unlabeled compound is at least 2 mass units.

25. The compound of claim 24, wherein the mass difference is between 3 and 15 mass units.

26. The compound of claim 1, wherein C is a quaternary nitrogen derivative.

27. The compound of claim 26, wherein the quaternary nitrogen comprises the formula:

28. The compound of claim 27, wherein R₅, and R₆ are methyl.

29. The compound of claim 28, wherein R₄ is methyl.

30. The compound of claim 28, wherein R₄ is tert-butyl.

31. A compound of formula:

32. The compound of claim 31, wherein X′

is NH, and R₄ is methyl or tert-butyl.

33. The compound of claim 32, wherein R′

is ethyl.

34. The compound of claim 32, wherein R is CH₂CH₂ or CH₂CH₂OCH₂CH₂.

35. The compound of claim 32, wherein X′

, R′ and

R are isotopically labeled.

36. The compound of claim 35, wherein R′

is CD₂CD₃ and R is CD₂CD₂, CH₂CH₂OCD₂CD₂, or CD₂CD₂OCD₂CD₂.

37. A compound of formula:

38. The compound of claim 37, wherein X′

is NH, and R₄ is methyl or tert-butyl.

39. The compound of claim 38, wherein R′

is ethyl.

40. The compound of claim 39, wherein R is CH₂CH₂ or CH₂CH₂OCH₂CH₂.

41. The compound of claim 37, wherein X′

, R′ and

R are isotopically labeled.

42. The compound of claim 41, wherein R′

is CD₂CD₃ and R is CD₂CD₂, CH₂CH₂OCD₂CD₂, or CD₂CD₂OCD₂CD₂.

43. A compound or formula:

44. The compound of claim 43, wherein X′

is NH, and R₄ is methyl or tert-butyl.

45. The compound of claim 44, wherein R′

is ethyl.

46. The compound of claim 44, wherein R is CH₂CH₂ or CH₂CH₂OCH₂CH₂.

47. The compound of claim 43, wherein X′

, R′ and

R are isotopically labeled.

48. The compound of claim 47, wherein R′

is CD₂CD₃ and R is CD₂CD₂, CH₂CH₂OCD₂CD₂, or CD₂CD₂OCD₂CD₂.

49. The compound of claim 43, wherein the peptide comprises a phosphoryl group.

50. A kit for detecting the presence of a plurality of target peptides in a sample, the kit comprising a compound of formula A-B-C wherein A is a nucleophilic reactive group, B is a detectable moiety capable of being isotopically labeled, and C is a charge replacement group;

and written instructions.

51. The kit of claim 50, wherein the compound comprises the formula:

52. The kit of claim 50, wherein the compound comprises the formula:

53. The kit of claim 50, further comprising means for separating a class of peptides.

54. The kit of claim 53, wherein the means for separating comprises an affinity column.

55. The kit of claim 50, further comprising buffers and protein digestion enzymes.