Protein sequencing via coupling of polymerizable molecules
Provided herein are 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 processing a peptide, comprising:
(a) providing said peptide, a capture moiety, 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 said 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 comprising an additional polymerizable molecule, 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.
2. The method of claim 1 , wherein said coupling of (g) is performed by coupling said polymerizable molecule of said linker to said additional polymerizable molecule of said additional linker.
3. The method of claim 1 , further comprising, cleaving said additional amino acid from said peptide.
4. The method of claim 1 , further comprising, translocating said stacked AALC complex through a nanopore or a nanogap.
5. The method of claim 4 , further comprising, identifying an amino acid type of said amino acid or said additional amino acid.
6. The method of claim 5 , wherein said identifying comprises measuring a signal as said stacked AALC complex translocates through said nanopore or said nanogap, thereby generating a measured signal, and using said measured signal to determine an identity of said amino acid or said additional amino acid.
7. The method of claim 5 , wherein said identifying comprises using imaging.
8. The method of claim 7 , wherein said identifying further comprises using a binding agent carrying a fluorophore or optically detectable tag, wherein said binding agent selectively binds to a particular amino acid type.
9. The method of claim 1 , wherein said capture moiety is coupled to a substrate.
10. The method of claim 1 , wherein said capture moiety is coupled to said peptide.
11. The method of claim 1 , wherein said capture moiety comprises a nucleic acid molecule.
12. The method of claim 1 , wherein said capture moiety comprises a pseudo-complementary base, a bridged nucleic acid, a xenonucleic acid, a locked nucleic acid, a peptide nucleic acid (PNA), a gamma-PNA, or a morpholino.
13. The method of claim 1 , wherein said capture moiety comprises a first nucleic acid molecule, wherein said polymerizable molecule 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.
14. The method of claim 13 , wherein said coupling of (c) is performed using a ligase.
15. The method of claim 14 , 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.
16. The method of claim 13 , wherein said coupling of (c) is performed by hybridizing at least a portion of said first nucleic acid molecule to at least a portion of said second nucleic acid molecule.
17. The method of claim 1 , wherein said capture moiety comprises a nucleic acid barcode molecule.
18. The method of claim 17 , wherein said nucleic acid barcode molecule identifies said peptide.
19. 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.
20. The method of claim 1 , wherein (b) occurs before (c).
21. The method of claim 1 , wherein (c) occurs before (b).
22. The method of claim 1 , further comprising, repeating (e)-(g) to generate said stacked AALC complex.
23. The method of claim 1 , wherein said polymerizable molecule comprises a nucleic acid barcode molecule that comprises temporal information.
24. The method of claim 23 , wherein said temporal information comprises a cycle barcode that indicates a round or iteration in which said polymerizable molecule is provided.
25. The method of claim 1 , wherein said capture moiety comprises a cleavable moiety.
26. The method of claim 25 , wherein said cleavable moiety is a restriction enzyme recognition site, an abasic site, a uracil, or a disulfide bond.
27. The method of claim 1 , wherein said cleaving of (d) is performed using treatment with acid.
28. The method of claim 27 , wherein said acid is a Lewis acid.
29. The method of claim 1 , wherein said amino acid comprises a post-translational modification or a non-standard amino acid.
30. The method of claim 1 , further comprising, (h) performing an extension reaction of said AALC complex or said stacked AALC complex, thereby generating an extension product, and (i) sequencing said extension product or a derivative thereof.