Protein sequencing via coupling of polymerizable molecules
The present disclosure provides methods and systems for sequencing proteins. One or more methods disclosed herein may use linkers having an amino acid-reactive group and an additional reactive moiety that may be used to couple a polymerizable molecule. The linker may couple to a polymerizable molecule and an amino acid of a peptide and a capture moiety via the polymerizable molecule, followed by cleavage of the amino acid from the peptide. Further processing and analysis may be conducted using, for example, nanopores or nanogaps.
1. A method of sequencing a peptide at single amino acid resolution, comprising:
(a) providing said peptide and a linker, wherein said linker comprises a polymerizable molecule, and wherein said linker is capable of coupling to an amino acid of said peptide;
(b) coupling said linker to said amino acid of said peptide;
(c) coupling said linker to a capture moiety;
(d) cleaving said amino acid from said peptide to yield an amino acid-linker-capture moiety (AALC) complex;
(e) providing an additional linker, wherein said additional linker is capable of coupling to an additional amino acid of said peptide;
(f) coupling said additional linker to said additional amino acid, thereby generating an additional amino acid-linker complex; and
(g) coupling said additional amino acid-linker complex to said AALC complex, thereby generating a stacked AALC complex;
(h) contacting said stacked AALC complex with a plurality of binding agents, wherein individual binding agents of said plurality of binding agents have different specificity to different amino acid types;
(i) translocating said stacked AALC complex contacted with said plurality of binding agents through a nanopore or a nanogap; and
(j) identifying an amino acid type of said amino acid or said additional amino acid.
2. The method of claim 1 , wherein said capture moiety is affixed to a substrate.
3. The method of claim 2 , wherein said substrate is substantially planar.
4. The method of claim 2 , wherein said substrate is a bead.
5. The method of claim 1 , wherein said capture moiety comprises a nucleic acid molecule.
6. The method of claim 5 , wherein said nucleic acid molecule comprises a DNA molecule, an RNA molecule, an XNA molecule, or modified variant thereof.
7. The method of claim 6 , wherein said DNA molecule is single-stranded.
8. The method of claim 1 , wherein said capture moiety comprises a first nucleic acid molecule, wherein said linker comprises a second nucleic acid molecule, and wherein said coupling of (c) comprises coupling said first nucleic acid molecule to said second nucleic acid molecule.
9. The method of claim 8 , wherein said coupling of (c) is performed using a ligase.
10. The method of claim 8 , wherein at least a portion of said first nucleic acid molecule is complementary to at least a portion of said second nucleic acid molecule.
11. The method of claim 10 , wherein said coupling is performed by hybridizing said at least said portion of said first nucleic acid molecule to said at least said portion of said second nucleic acid molecule.
12. The method of claim 8 , wherein said coupling is performed using a splint oligonucleotide, wherein said splint oligonucleotide comprises a first sequence complementary to at least a portion of said first nucleic acid molecule and a second sequence complementary to at least a portion of said second nucleic acid molecule.
13. The method of claim 1 , wherein said capture moiety comprises a nucleic acid barcode molecule.
14. The method of claim 13 , wherein said nucleic acid barcode molecule identifies said peptide.
15. The method of claim 1 , wherein said capture moiety is coupled to said peptide.
16. The method of claim 1 , wherein said amino acid or said additional amino acid is an N-terminal amino acid or a C-terminal amino acid.
17. The method of claim 1 , wherein (b) occurs before (c).
18. The method of claim 1 , wherein (c) occurs before (b).
19. The method of claim 1 , further comprising, repeating (e)-(g) to generate said stacked AALC complex.
20. The method of claim 1 , wherein said identifying further comprises measuring a signal as said stacked AALC complex contacted with said plurality of binding agents translocates through said nanopore or said nanogap.
21. The method of claim 1 , wherein said translocating of (i) occurs at a temperature below ambient temperature.
22. The method of claim 1 , wherein said linker comprises a nucleic acid barcode molecule that comprises temporal information.
23. The method of claim 1 , wherein said capture moiety comprises a cleavable moiety.
24. The method of claim 1 , wherein said nanopore or said nanogap is a solid-state nanopore.
25. The method of claim 1 , wherein said plurality of binding agents comprises an antibody, antibody fragment, nanobody, or aptamer.
26. The method of claim 1 , wherein said cleaving of (d) is performed using treatment with acid.
27. The method of claim 26 , wherein said acid is a Lewis acid.
28. The method of claim 1 , wherein said amino acid comprises a post-translational modification or a non-standard amino acid, and wherein (j) comprises identifying said post-translational modification or said non-standard amino acid.
29. The method of claim 1 , wherein (a)-(g) are performed in absence of a substrate.