Methods and compositions for protein sequencing
Aspects of the application provide methods of identifying and sequencing proteins, polypeptides, and amino acids, and compositions useful for the same. In some aspects, the application provides methods of obtaining data during a degradation process of a polypeptide, and outputting a sequence representative of the polypeptide. In some aspects, the application provides amino acid recognition molecules comprising a shielding element that enhances photostability in polypeptide sequencing reactions.
1. A method of sequencing a polypeptide, the method comprising:
contacting a single polypeptide molecule in a reaction mixture with a composition comprising one or more terminal amino acid recognition molecules and a cleaving reagent, wherein the single polypeptide molecule is immobilized to a surface through a linker comprising an oligonucleotide; and
detecting a series of signal pulses indicative of association of the one or more terminal amino acid recognition molecules with a terminus of the single polypeptide molecule in the presence of the cleaving reagent, wherein the series of signal pulses is indicative of a series of amino acids exposed at the terminus over time as a result of terminal amino acid cleavage by the cleaving reagent.
2. The method of claim 1 , wherein association of the one or more terminal amino acid recognition molecules with each type of amino acid exposed at the terminus produces a characteristic pattern in the series of signal pulses that is different from other types of amino acids exposed at the terminus.
3. The method of claim 2 , wherein the characteristic pattern comprises a portion of the series of signal pulses.
4. The method of claim 3 , wherein a signal pulse of the characteristic pattern corresponds to an individual association event between a terminal amino acid recognition molecule and an amino acid exposed at the terminus.
5. The method of claim 4 , wherein the signal pulse of the characteristic pattern comprises a pulse duration that is characteristic of a dissociation rate of binding between the terminal amino acid recognition molecule and the amino acid exposed at the terminus.
6. The method of claim 5 , wherein each signal pulse of the characteristic pattern is separated from another by an interpulse duration that is characteristic of an association rate of terminal amino acid recognition molecule binding.
7. The method of claim 2 , wherein the characteristic pattern corresponds to a series of reversible terminal amino acid recognition molecule binding interactions with the amino acid exposed at the terminus of the single polypeptide molecule.
8. The method of claim 7 , wherein the series of reversible terminal amino acid recognition molecule binding interactions comprises a reversible formation of one binary complex species at the terminus of the single polypeptide molecule.
9. The method of claim 2 , wherein the characteristic pattern is indicative of the amino acid exposed at the terminus of the single polypeptide molecule and an amino acid at a contiguous position.
10. The method of claim 1 , wherein sequencing comprises identifying at least a portion of all types of successive amino acids exposed at the terminus of the single polypeptide while the single polypeptide is being degraded by the cleaving reagent.
11. The method of claim 1 , wherein each of the one or more terminal amino acid recognition molecules comprises a recognition protein or a nucleic acid aptamer.
12. The method of claim 11 , wherein the recognition protein is a degradation pathway protein, a peptidase, an antibody, an aminotransferase, a tRNA synthetase, or an SH2 domain-containing protein or fragment thereof.
13. The method of claim 1 , wherein each of the one or more terminal amino acid recognition molecules comprises a detectable label.
14. The method of claim 13 , wherein the detectable label is a luminescent label or a conductivity label.
15. The method of claim 1 , wherein the series of signal pulses is a series of real-time signal pulses.
16. A system comprising:
at least one hardware processor; and
at least one non-transitory computer-readable storage medium storing processor-executable instructions that, when executed by the at least one hardware processor, cause the at least one hardware processor to perform the method of claim 1 .
17. At least one non-transitory computer-readable storage medium storing processor-executable instructions that, when executed by at least one hardware processor, cause the at least one hardware processor to perform the method of claim 1 .
18. The method of claim 2 , wherein signal pulses of the characteristic pattern comprise a mean pulse duration of between about 1 millisecond and about 10 seconds.
19. The method of claim 18 , wherein the mean pulse duration is between about 10 milliseconds and about 100 milliseconds or between about 100 milliseconds and about 500 milliseconds.
20. The method of claim 2 , wherein the characteristic pattern of one type of amino acid is different from the characteristic pattern of another type of amino acid by a mean pulse duration of at least 10 milliseconds.
21. The method of claim 20 , wherein the characteristic pattern of one type of amino acid is different from the characteristic pattern of another type of amino acid by a mean pulse duration of between about 10 milliseconds and about 100 milliseconds or between about 100 milliseconds and about 10 seconds.
22. The method of claim 1 , wherein the reaction mixture comprises a terminal amino acid recognition molecule and the cleaving reagent in a ratio of between about 10:1 and about 200:1.
23. The method of claim 1 , wherein the reaction mixture comprises a terminal amino acid recognition molecule and the cleaving reagent in a ratio of between about 50:1 and about 150:1.
24. The method of claim 1 , wherein the surface is a surface of a sample well.
25. The method of claim 1 , wherein the oligonucleotide is a DNA oligonucleotide.
26. The method of claim 1 , wherein the linker further comprises a peptide and/or a polyethylene glycol (PEG).
27. The method of claim 1 , wherein the linker further comprises a biotin molecule or an avidin protein.