Claims
- 1. A polypeptide having a modified lysine residue of Formula I:
- 2. The polypeptide having a modified lysine residue according to claim 1, wherein said optionally substituted monocyclic or bicyclic ring system is selected from the group consisting of imidozolinyl, imidazolindinyl, pyrimidinyl, imidazolyl, purinyl, quanazolinyl and pteridinyl.
- 3. The polypeptide having a modified lysine residue according to claim 1, said polypeptide having Formula Ia:
- 4. The polypeptide having a modified lysine residue according to claim 1, said polypeptide having Formula Ib:
- 5. The polypeptide having a modified lysine residue according to claim 1, wherein R1, R2, R3, R4 and R5 are each hydrogen.
- 6. The polypeptide having a modified lysine residue according to claim 1, wherein R1, R2, R3, R4 and R5 are each deuterium.
- 7. The polypeptide having a modified lysine residue according to claim 1, wherein R1 and R2 are each alkyl.
- 8. The polypeptide having a modified lysine residue according to claim 1, wherein R1 and R2 are each independently a member selected from the group consisting of methyl, ethyl, propyl and butyl.
- 9. A compound of Formula II:
- 10. The compound of claim 9, wherein said LG is selected from the group consisting of a halogen, an alkoxy group, an alkylthio group, an aryloxy group, a tosyl group and an arylthio group.
- 11. The compound of claim 9, wherein said compound is of the formula IIa
- 12. The compound of claim 11, wherein said compound is of the formula IIb:
- 13. The compound according to claim 12, wherein R1, R2, R3, R4 and R5 are each hydrogen.
- 14. The compound according to claim 12, wherein R1, R2, R3, R4 and R5 are each deuterium.
- 15. The compound according to claim 12, wherein R1 and R2 are each alkyl.
- 16. The compound according to claim 12, wherein R1 and R2 arc each independently a member selected from the group consisting of methyl, ethyl, propyl and butyl.
- 17. The compound according to claim 12, wherein R2 and R3 and the carbons to which they are attached join to form a 6-member carbocyclic ring.
- 18. The compound according to claim 12, wherein R5 is a methyl group.
- 19. The compound according to claim 12, wherein R5 is benzoic acid.
- 20. The compound according to claim 12, wherein R1 comprises an affinity tag.
- 21. A method of derivatizing a polypeptide having a lysine residue, said method comprising:
incubating said polypeptide having a lysine residue with a compound of Formula II to form a polypeptide with a lysine residue of Formula I: 27wherein each R is independently a member selected from the group consisting of hydrogen, deuterium, halogen, hydroxyl, cyano, optionally substituted alkyl, optionally substituted alkylcarbamoyl, optionally substituted alkoxy, optionally substituted alkoxycarbonyl, optionally substituted aryl, optionally substituted aryloxy, optionally substituted aryloxycarbonyl, optionally substituted arylcarbamoyl, optionally substituted siloxanly and an affinity tag; mis0-7;and wherein the circle joining the two nitrogens represents an optionally substituted monocyclic or bicyclic ring system having between 2 and 12 additional ring atoms, and wherein said ring atoms are each selected from the group consisting of carbon, oxygen, nitrogen, sulfur and silicon, thereby derivatizing said polypeptide having a lysine residue.
- 22. The method of derivatizing a polypeptide according to claim 21, wherein said polypeptide has Formula Ia:
- 23. The method of derivatizing a polypeptide according to claim 22, wherein said polypeptide has Formula Ib:
- 24. The method of derivatizing a polypeptide according to claim 22, wherein R1, R2, R3, R4 and R5 are each hydrogen.
- 25. The method of derivatizing a polypeptide according to claim 22, wherein each of R1, R2, R3 and R4 are each deuterium.
- 26. The method of derivatizing a polypeptide according to claim 22, wherein R1 and R2 are each alkyl.
- 27. The method of derivatizing a polypeptide according to claim 22, wherein R1 and R2 are each independently a member selected from the group consisting of methyl, ethyl, propyl and butyl.
- 28. The method of derivatizing a polypeptide according to claim 22, wherein R1 is an affinity tag.
- 29. A method for mass spectrometric polypeptide analysis, said method comprising:
a) ionizing a modified polypeptide having a lysine residue of Formula I: 30wherein each R is independently a member selected from the group consisting of hydrogen, deuterium, halogen, hydroxyl, cyano, optionally substituted alkyl, optionally substituted alkylcarbamoyl, optionally substituted alkoxy, optionally substituted alkoxycarbonyl, optionally substituted aryl, optionally substituted aryloxy, optionally substituted aryloxycarbonyl, optionally substituted arylcarbamoyl, optionally substituted siloxanyl, and an affinity tag; mis0-7;and wherein the circle joining the two nitrogens represents an optionally substituted monocyclic or bicyclic ring system having between 2 and 12 additional ring atoms, and wherein said ring atoms are each selected from the group consisting of carbon, oxygen, nitrogen, sulfur and silicon; and b) analyzing the results of the ionized modified polypeptide.
- 30. The method for mass spectrometric polypeptide analysis according to claim 29, wherein said modified polypeptide having a lysine residue of Formula I has Formula Ia:
- 31. The method for mass spectrometric polypeptide analysis according to claim 30, wherein said modified polypeptide having a lysine residue of Formula I has Formula lb
- 32. The method for mass spectrometric polypeptide analysis according to claim 30, wherein R1, R2, R3 and R4 are each hydrogen.
- 33. The method for mass spectrometric polypeptide analysis according to claim 30, wherein R1, R2, R3 and R4 are each deuterium.
- 34. The method for mass spectrometric polypeptide analysis according to claim 30, wherein R1 and R2 are each alkyl.
- 35. The method for mass spectrometric polypeptide analysis according to claim 30, further comprising enzymatically digesting said modified polypeptide prior to step (a).
- 36. The method for mass spectrometric polypeptide analysis according to claim 30, wherein R1 is an affinity tag, and said method further comprises purifying said modified polypeptide using said affinity tag prior to step (a).
- 37. The method for mass spectrometric polypeptide analysis according to claim 30, wherein the ionization of step (a) comprises matrix-assisted desorption/ionization mass spectrometry.
- 38. The method for mass spectrometric polypeptide analysis according to claim 30, wherein the ionization of step (a) comprises electrospray ionization mass spectrometry.
- 39. A method for analyzing a sequentially labeled polypeptide, said method comprising:
incubating said polypeptide with a first label to form a first labeled polypeptide; incubating said first labeled polypeptide with a second label to form a second labeled polypeptide, wherein at least one of said first label or said second label is a compound having Formula II: 33wherein each R is independently a member selected from the group consisting of hydrogen, deuterium, halogen, hydroxyl, cyano, optionally substituted alkyl, optionally substituted alkylcarbamoyl, optionally substituted alkoxy, optionally substituted alkoxycarbonyl, optionally substituted aryl, optionally substituted aryloxy, optionally substituted aryloxycarbonyl, optionally substituted arylcarbamoyl and an affinity tag; mis0-7; wherein the circle joining the two nitrogens represents an optionally substituted monocyclic or bicyclic ring system having between 2 and 12 additional ring atoms, and wherein said ring atoms are each selected from the group consisting of carbon, oxygen, nitrogen, sulfur and silicon; LG is a leaving group; and analyzing said second labeled polypeptide with a mass spectrometer.
- 40. The method for analyzing a sequentially labeled polypeptide according to claim 39, wherein said compound having Formula II, has Formula Ia
- 41. The method for analyzing a sequentially labeled polypeptide according to claim 40, further comprising labeling said second labeled polypeptide with a third label to form a third labeled polypeptide.
- 42. The method for analyzing a sequentially labeled polypeptide according to claim 40, wherein said first label, said second label and said third label are each independently selected from the group consisting of a compound of Formula II, and an amino acid specific label.
- 43. The method for analyzing a sequentially labeled polypeptide according to claim 42, wherein said amino acid specific label is a member selected from the group consisting of a cysteine labeling reagent, an arginine label reagent and a carboxylic acid labeling reagent.
- 44. The method for analyzing a sequentially labeled polypeptide according to claim 42, wherein said carboxylic acid reagent is an alcohol under esterifying conditions to form an ester.
- 45. The method for analyzing a sequentially labeled polypeptide according to claim 42, wherein said high mass accuracy mass spectrometer is a Fourier transform ion cyclotron resonance spectrometer.
- 46. A method for increasing the ionization efficiency of a lysine-containing polypeptide, said method comprising:
a) incubating a lysine-containing polypeptide with a compound of Formula II to form a modified polypeptide having a lysine residue of Formula I: 35wherein each R is independently a member selected from the group consisting of hydrogen, deuterium, halogen, hydroxyl, cyano, optionally substituted alkyl, optionally substituted alkylcarbamoyl, optionally substituted alkoxy, optionally substituted alkoxycarbonyl, optionally substituted aryl, optionally substituted aryloxy, optionally substituted aryloxycarbonyl, optionally substituted arylcarbamoyl and an affinity tag; mis0-7;and wherein the circle joining the two nitrogens represents an optionally substituted monocyclic or bicyclic ring system having between 2 and 12 additional ring atoms, and wherein said ring atoms are each selected from the group consisting of carbon, oxygen, nitrogen, sulfir and silicon. and b) ionizing said modified polypeptide having a lysine residue of Formula I, thereby increasing the ionization efficiency of said lysine-containing polypeptide.
- 47. The method for increasing the ionization efficiency of a lysine-containing polypeptide according to claim 46, wherein said compound of Formula I has Formula Ia:
- 48. The method for increasing the ionization efficiency of a lysine-containing polypeptide according to claim 47, wherein said compound of Formula I has Formula Ib:
- 49. A method for performing differential quantitation of a lysine-containing polypeptide, said method comprising:
a) incubating a first sample of a lysine-containing polypeptide with a first isotopic version of a compound of Formula II to form a first modified polypeptide having a lysine residue of Formula I: 38wherein each R is independently a member selected from the group consisting of hydrogen, deuterium, halogen, hydroxyl, cyano, optionally substituted alkyl, optionally substituted alkylcarbamoyl, optionally substituted alkoxy, optionally substituted alkoxycarbonyl, optionally substituted aryl, optionally substituted aryloxy, optionally substituted aryloxycarbonyl, optionally substituted arylcarbamoyl, optionally substituted siloxanyl and an affinity tag; m is 0-7; and wherein the circle joining the two nitrogens represents an optionally substituted monocyclic or bicyclic ring system having between 2 and 12 additional ring atoms, and wherein said ring atoms are each selected from the group consisting of carbon, oxygen, nitrogen, sulfur and silicon; b) incubating a second sample of said lysine-containing polypeptide with a second isotopic version of a compound of Formula II to form a second modified polypeptide having a lysine residue of Formula I; c) combining said first sample and said second sample of said modified polypeptide having a lysine residue of Formula I to form a mixture; and d) ionizing said mixture to form a series of isotopically labeled polypeptide pairs, wherein the relative concentrations of said first modified polypeptide having a lysine residue of Formula I and said second modified polypeptide having a lysine residue of Formula I are directly proportional to their relative signal intensities, and wherein the mass difference between the two modified polypeptides indicates the number of lysine(s) that are present in said lysine-containing polypeptide.
- 50. The method for performing differential quantitation of a lysine-containing polypeptide according to claim 49, wherein said series of isotopically labeled polypeptide pairs indicates that at least two lysines are present.
- 51. The method for performing differential quantitation of a lysine-containing polypeptide according to claim 49, wherein said first sample and said second sample of said lysine-containing polypeptides are combined in equal proportions.
- 52. A method for determining the number of lysines in a lysine-containing polypeptide, said method comprising:
a) incubating a sample of a lysine-containing polypeptide with a first isotopic version of a compound of Formula II and a second isotopic version of a compound of Formula II to form a mixture having a first modified polypeptide having a lysine residue of Formula I and a second modified polypeptide having a lysine residue of Formula I, wherein Formula I has the formula: 39wherein each R is independently a member selected from the group consisting of hydrogen, deuterium, halogen, hydroxyl, cyano, optionally substituted alkyl, optionally substituted alkylcarbamoyl, optionally substituted alkoxy, optionally substituted alkoxycarbonyl, optionally substituted aryl, optionally substituted aryloxy, optionally substituted aryloxycarbonyl, optionally substituted arylcarbamoyl, optionally substituted siloxanyl and an affinity tag; mis0-7;and wherein the circle joining the two nitrogens represents an optionally substituted monocyclic or bicyclic ring system having between 2 and 12 additional ring atoms, and wherein said ring atoms are each selected from the group consisting of carbon, oxygen, nitrogen, sulfur and silicon; and b) ionizing said mixture to form a series of isotopically labeled polypeptide pairs, wherein the mass difference between said first modified polypeptide having a lysine residue of Formula I and said second modified polypeptide having a lysine residue of Formula I indicates the number of lysine(s) that are present in said lysine-containing polypeptide.
- 53. The method for determining the number of lysines in a lysine-containing polypeptide according to 52, wherein said series of isotopically labeled polypeptide pairs indicates that at least two lysines are present.
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application claims priority to U.S. Provisional Application Nos. 60/332,988, filed Nov. 5, 2001, 60/385,835, filed Jun. 3, 2002 and 60/410,382, filed Sep. 12, 2002, the teachings of each of which are hereby incorporated by reference in their entirety for all purposes.
Provisional Applications (3)
|
Number |
Date |
Country |
|
60410382 |
Sep 2002 |
US |
|
60385835 |
Jun 2002 |
US |
|
60332988 |
Nov 2001 |
US |