Single-molecule protein and peptide sequencing

Abstract

This specification provides methods, assays, and reagents useful for sequencing proteins. Protein sequencing in a broad sense involves observing valid amino acid identity and order, which is useful for sequencing a single polypeptide molecule or multiple molecules of a single polypeptide. In one embodiment, this method is useful for sequencing multiple polypeptides. The methods and reagents described herein may be useful for high-resolution investigations of the proteome and may enable ultra-sensitive diagnostics, which are important for the early detection of diseases. [Solution] A method for identifying the terminal amino acids of a peptide, (a) A step of contacting the peptide with a ClickP compound, wherein the ClickP compound binds to the terminal amino acids or terminal amino acid derivatives of the peptide to form a ClickP peptide complex, (b) A step of attaching the ClickP peptide complex to a substrate, (c) A step of cleaving the complex from the peptide, thereby providing the ClickP amino acid complex, (d) A method comprising the step of detecting the ClickP amino acid complex.

Claims

1. A method for identifying the terminal amino acids of a peptide, (a) A step of contacting the peptide with a ClickP compound, wherein the ClickP compound binds to the terminal amino acid or terminal amino acid derivative of the peptide to form a ClickP peptide complex; (b) A step of attaching the ClickP peptide complex to a substrate, (c) A step of cleaving the complex from the peptide, thereby providing the ClickP amino acid complex, (d) A method comprising the step of detecting the ClickP amino acid complex.

2. The method according to claim 1, wherein the step of detecting the ClickP amino acid complex includes a step of contacting the ClickP amino acid complex with one or more ClickP amino acid complex binders, wherein the ClickP amino acid complex binders bind to the ClickP amino acid complex or a subgroup of the ClickP amino acid complex.

3. The method according to claim 1, wherein the step of detecting the ClickP amino acid complex includes direct detection using the wavelength of light.

4. The method according to any one of claims 1 to 3, further comprising the step of identifying the amino acids of the ClickP amino acid complex.

5. The method according to any one of claims 1 to 4, wherein the ClickP compound binds to the N-terminal amino acid or N-terminal amino acid derivative of the peptide to form a ClickP amino acid complex.

6. The method according to any one of claims 1 to 4, wherein the ClickP compound binds to the C-terminal amino acid or C-terminal amino acid derivative of the peptide to form a ClickP amino acid complex.

7. The method according to any one of claims 1 to 6, further comprising the step (e) of releasing the ClickP amino acid complex from the substrate.

8. The method according to any one of claims 1 to 7, wherein steps (a) to (e) are repeated.

9. The method according to any one of claims 1 to 8, wherein any excess and / or unbound ClickP compound is washed away before step (b) or step (c).

10. The method according to any one of claims 1 to 9, wherein the peptide is attached to the substrate.

11. The method according to claim 10, wherein the peptide is attached to the substrate via the C'-terminal carboxyl group or side-chain functional group of the peptide.

12. The method according to claim 10, wherein the peptide is attached to a substrate via the N'-terminal amino group or side-chain functional group of the polypeptide.

13. The method according to claim 10, wherein the peptide is covalently attached to the substrate.

14. The method according to any one of claims 1 to 13, wherein the substrate is optically transparent.

15. The method according to any one of claims 1 to 14, wherein the substrate includes a functionalized surface.

16. The method according to claim 15, wherein the functionalized surface is selected from the group consisting of an azide functionalized surface, a thiol functionalized surface, an alkyne, DBCO, maleimide, succinimide, tetrazine, TCO, vinyl, methylcyclopropene, a primary amine surface, a carboxyl surface, a DBCO surface, an alkyne surface, and an aldehyde surface.

17. The method according to any one of claims 1 to 16, wherein the substrate includes a plurality of bonding points.

18. The method according to claim 17, wherein the peptide is attached to the binding site.

19. The one or more ClickP amino acid complex binders are (a) One or more binders that bind to a subgroup of 20 natural proteinogenic amino acids that form a complex with ClickP, (b) One or more binders that bind to a subgroup of post-translationally modified amino acids that form a complex with ClickP, (c) One or more binders bonded to a derivative of (a) or (b), The method of claim 2, comprising (d) a combination of the binders of (a), (b), or (c).

20. The one or more ClickP amino acid complex binders are (a) One or more binders that bind to one of 20 types of naturally occurring proteinogenic amino acids that form a complex with ClickP, (b) One or more binders that bind to a post-translationally modified amino acid that forms a complex with ClickP, (c) One or more binders bonded to a derivative of (a) or (b), The method of claim 2, comprising (d) a combination of the binders of (a), (b), or (c).

21. The method according to claim 19 or 20, wherein at least one binder includes a detectable label.

22. The method according to claim 21, wherein the step of detecting the binder bound to the ClickP amino acid complex includes detecting the detectable label.

23. The method according to claim 1, further comprising a detectable label for the ClickP compound.

24. A method for identifying the terminal amino acids of two or more peptides in a sample, (a) A step of independently attaching the two or more peptides to binding sites on a substrate, (b) A step of contacting the peptide with a ClickP compound, wherein the ClickP compound binds to a terminal amino acid or terminal amino acid derivative to form a ClickP peptide complex, (c) A step of attaching the ClickP peptide complex to the substrate, (d) A step of cleaving the ClickP peptide complex from the peptide, thereby providing the ClickP amino acid complex, (e) A method comprising the step of detecting the ClickP amino acid complex.

25. The method according to claim 24, wherein the step of detecting the ClickP amino acid complex includes a step of contacting the ClickP amino acid complex with one or more ClickP amino acid complex binders, wherein the ClickP amino acid complex binders bind to the ClickP amino acid complex or a subgroup of the ClickP amino acid complex.

26. The method according to claim 24, wherein the step of detecting the ClickP amino acid complex includes direct detection using the wavelength of light.

27. The method according to any one of claims 1 to 26, further comprising the step of identifying the amino acids of the ClickP amino acid complex.

28. The method according to any one of claims 24 to 27, wherein the ClickP compound binds to the N-terminal amino acid or N-terminal amino acid derivative of the peptide to form a ClickP amino acid complex.

29. The method according to any one of claims 24 to 27, wherein the ClickP compound binds to the C-terminal amino acid or C-terminal amino acid derivative of the peptide to form a ClickP amino acid complex.

30. The method according to any one of claims 24 to 29, further comprising the step (f) of releasing the ClickP amino acid complex from the substrate.

31. The method according to any one of claims 24 to 30, wherein steps (b) to (f) are repeated.

32. The method according to any one of claims 24 to 31, wherein any excess and / or unbound ClickP compound is washed away before step (c) or step (d).

33. The method according to claim 24, wherein one or more peptides are attached to the substrate via the C'-terminal carboxyl group or side-chain functional group of a polypeptide or protein.

34. The method according to claim 24, wherein one or more peptides are attached to a substrate via the N'-terminal carboxyl group or side-chain functional group of the peptides.

35. The method according to any one of claims 24 to 34, wherein one or more peptides are covalently attached to the substrate.

36. The method according to any one of claims 24 to 35, wherein the substrate is optically transparent.

37. The method according to any one of claims 24 to 36, wherein the substrate includes a functionalized surface.

38. The method according to claim 37, wherein the functionalized surface is selected from the group consisting of an azide functionalized surface, a thiol functionalized surface, an alkyne, DBCO, maleimide, succinimide, tetrazine, TCO, vinyl, methylcyclopropene, a primary amine surface, a carboxyl surface, a DBCO surface, an alkyne surface, and an aldehyde surface.

39. The one or more ClickP amino acid complex binders are (a) One or more binders that bind to a subgroup of 20 natural proteinogenic amino acids that form a complex with ClickP, (b) One or more binders that bind to a subgroup of post-translationally modified amino acids that form a complex with ClickP, (c) One or more binders bonded to a derivative of (a) or (b), The method of claim 25, comprising (d) a combination of the binders of (a), (b), or (c).

40. The one or more ClickP amino acid complex binders are (a) One or more binders that bind to one of 20 types of naturally occurring proteinogenic amino acids that form a complex with ClickP, (b) One or more binders that bind to a post-translationally modified amino acid that forms a complex with ClickP, (c) One or more binders bonded to a derivative of (a) or (b), The method of claim 25, comprising (d) a combination of the binders of (a), (b), or (c).

41. The method according to claim 39 or 40, wherein at least one binder includes a detectable label.

42. The method according to claim 41, wherein the step of detecting the binder bound to the ClickP amino acid complex includes detecting the detectable label.

43. The method according to claim 24, further comprising a detectable label for the ClickP compound.

44. The method according to any one of claims 24 to 43, wherein the sample comprises a biological fluid, a cell extract, or a tissue extract.

45. A method for sequencing at least a portion of a peptide, (a) A step of contacting the peptide with a ClickP compound, wherein the ClickP compound binds to the terminal amino acid or terminal amino acid derivative of the peptide to form a ClickP peptide complex, (b) A step of attaching the ClickP peptide complex to a substrate, (c) A step of cleaving the ClickP peptide complex from the peptide to form a ClickP amino acid complex, (d) A step of detecting the ClickP amino acid complex, (e) A step of identifying the amino acids of the ClickP amino acid complex, (f) A step of releasing the ClickP amino acid complex from the substrate, (g) A method comprising a step of repeating steps (a) to (f).

46. The method according to claim 45, wherein the step of detecting the ClickP amino acid complex includes a step of contacting the ClickP amino acid complex with one or more ClickP amino acid complex binders, wherein the ClickP amino acid complex binders bind to the ClickP amino acid complex or a subgroup of the ClickP amino acid complex.

47. The method according to claim 45, wherein the step of detecting the ClickP amino acid complex includes direct detection using the wavelength of light.

48. The method according to any one of claims 45 to 47, wherein the ClickP compound binds to the N-terminal amino acid or N-terminal amino acid derivative of the peptide to form a ClickP amino acid complex.

49. The method according to any one of claims 45 to 47, wherein the ClickP compound binds to the C-terminal amino acid or C-terminal amino acid derivative of the peptide to form a ClickP amino acid complex.

50. The method according to any one of claims 45 to 49, wherein any excess and / or unbound ClickP compound is washed away before step (b) or step (c).

51. The method according to any one of claims 45 to 50, wherein the peptide is attached to the substrate.

52. The method according to claim 51, wherein the peptide is attached to a substrate via the C'-terminal carboxyl group or side-chain functional group of the peptide.

53. The method according to claim 51, wherein the peptide is attached to a substrate via the N'-terminal carboxyl group or side-chain functional group of the peptide.

54. The method according to any one of claims 45 to 53, wherein the peptide is covalently attached to the substrate.

55. The method according to any one of claims 45 to 54, wherein the substrate is optically transparent.

56. The method according to any one of claims 45 to 55, wherein the substrate includes a functionalized surface.

57. The method according to claim 56, wherein the functionalized surface is selected from the group consisting of an azide functionalized surface, a thiol functionalized surface, an alkyne, DBCO, maleimide, succinimide, tetrazine, TCO, vinyl, methylcyclopropene, a primary amine surface, a carboxyl surface, a DBCO surface, an alkyne surface, and an aldehyde surface.

58. The method according to any one of claims 45 to 57, wherein the substrate includes one or more bonding points.

59. The method according to claim 58, wherein the peptide is attached to the binding site.

60. The one or more ClickP amino acid complex binders are (a) One or more binders that bind to a subgroup of 20 natural proteinogenic amino acids that form a complex with ClickP, (b) One or more binders that bind to a subgroup of post-translationally modified amino acids that form a complex with ClickP, (c) One or more binders bonded to a derivative of (a) or (b), The method according to claim 46, comprising (d) a combination of the binders of (a), (b), or (c).

61. The one or more ClickP amino acid complex binders are (a) One or more binders that bind to one of 20 types of naturally occurring proteinogenic amino acids that form a complex with ClickP, (b) One or more binders that bind to a post-translationally modified amino acid that forms a complex with ClickP, (c) One or more binders bonded to a derivative of (a) or (b), The method according to claim 46, comprising (d) a combination of the binders of (a), (b), or (c).

62. The method according to claim 60 or 61, wherein at least one binder includes a detectable label.

63. The method according to claim 62, wherein the step of detecting the binder bound to the ClickP amino acid complex includes detecting the detectable label.

64. The method according to claim 45, further comprising a detectable label for the ClickP compound.

65. The method according to claim 45, further comprising comparing the sequence of the peptide determined in step (g) with a reference protein sequence database.

66. A method for sequencing at least a portion of two or more peptides in a sample, (a) A step of independently attaching the two or more peptides to binding sites on a substrate, (b) A step of contacting two or more peptides with a ClickP compound, wherein the ClickP compound binds to a terminal amino acid or terminal amino acid derivative to form a ClickP peptide complex, (c) A step of attaching the ClickP peptide complex to the substrate, (d) A step of cleaving the ClickP peptide complex from the peptide to form a ClickP amino acid complex, (e) A step of detecting the ClickP amino acid complex, (f) A step of identifying the amino acids of the ClickP amino acid complex, (g) A step of releasing the ClickP amino acid complex from the substrate, A method comprising (h) a process of repeating steps (b) to (g).

67. The method according to claim 66, wherein the step of detecting the ClickP amino acid complex includes a step of contacting the ClickP amino acid complex with one or more ClickP amino acid complex binders, wherein the ClickP amino acid complex binders bind to the ClickP amino acid complex or a subgroup of the ClickP amino acid complex.

68. The method according to claim 66, wherein the step of detecting the ClickP amino acid complex includes direct detection using the wavelength of light.

69. The method according to any one of claims 66 to 68, wherein the ClickP compound binds to the N-terminal amino acid or N-terminal amino acid derivative of the peptide to form a ClickP amino acid complex.

70. The method according to any one of claims 66 to 68, wherein the ClickP compound binds to the C-terminal amino acid or C-terminal amino acid derivative of the peptide to form a ClickP amino acid complex.

71. The method according to any one of claims 66 to 70, wherein any excess and / or unbound ClickP compound is washed away before step (c) or step (d).

72. The method according to claim 66, wherein the plurality of peptides are attached to the substrate via the C'-terminal carboxyl group or side-chain functional group of the peptides.

73. The method according to claim 66, wherein the plurality of peptides are attached to the substrate via the N'-terminal carboxyl group or side-chain functional group of the polypeptide.

74. The method according to any one of claims 66 to 73, wherein the two or more peptides are covalently attached to the substrate.

75. The method according to any one of claims 66 to 74, wherein the substrate is optically transparent.

76. The method according to any one of claims 66 to 74, wherein the substrate includes a functionalized surface.

77. The method according to claim 76, wherein the functionalized surface is selected from the group consisting of an azide functionalized surface, a thiol functionalized surface, an alkyne, DBCO, maleimide, succinimide, tetrazine, TCO, vinyl, methylcyclopropene, a primary amine surface, a carboxyl surface, a DBCO surface, an alkyne surface, and an aldehyde surface.

78. The ClickP amino acid complex binder is (a) One or more binders that bind to a subgroup of 20 natural proteinogenic amino acids that form a complex with ClickP, (b) One or more binders that bind to a subgroup of post-translationally modified amino acids that form a complex with ClickP, (c) One or more binders bonded to a derivative of (a) or (b), The method according to claim 67, comprising (d) a combination of the binders of (a), (b), or (c).

79. The ClickP amino acid complex binder is (a) One or more binders that bind to one of 20 types of naturally occurring proteinogenic amino acids that form a complex with ClickP, (b) One or more binders that bind to a post-translationally modified amino acid that forms a complex with ClickP, (c) One or more binders bonded to a derivative of (a) or (b), The method according to claim 67, comprising (d) a combination of the binders of (a), (b), or (c).

80. The method according to claim 78 or 79, wherein at least one binder includes a detectable label.

81. The method according to claim 80, wherein the step of detecting the binder bound to the ClickP amino acid complex includes detecting the detectable label.

82. The method according to claim 66, further comprising a detectable label for the ClickP compound.

83. The method according to any one of claims 66 to 82, wherein the sample comprises a mixture of biological fluid, cell extract, tissue extract, or synthetically synthesized peptides.

84. The method according to any one of claims 66 to 83, further comprising comparing the sequence of at least one peptide determined in step (h) with a reference protein sequence database.

85. The method according to any one of claims 66 to 83, further comprising comparing the sequences of each peptide determined in step (h), classifying similar peptide sequences, and counting a number of examples of each similar peptide sequence.

86. ClickP amino acid complex, (a) ClickP compounds that are bound to one of 20 types of naturally occurring proteinogenic amino acids, (b) ClickP compounds that bind to post-translationally modified amino acids, or (c) A ClickP amino acid complex comprising a ClickP compound bound to a derivative of (a) or (b).

87. ClickP amino acid complex binder, (a) A binder that binds to a subgroup of 20 natural proteinogenic amino acids that form a complex with ClickP. (b) A binder that binds to a subgroup of post-translationally modified amino acids that form a complex with ClickP, or (c) A ClickP amino acid complex binder comprising a binder that binds to a derivative of (a) or (b).

88. ClickP amino acid complex binder, (a) A binder that binds to one of the 20 natural proteinogenic amino acids that form a complex with ClickP, (b) A binder that binds to a post-translationally modified amino acid that forms a complex with ClickP, or (c) A ClickP amino acid complex binder comprising a binder that binds to a derivative of (a) or (b).

89. The binder according to claim 87 or 88, further comprising a detectable label.

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