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 - 272 . (canceled)
273 . A method of determining an amino acid sequence of a polypeptide, the method comprising:
i. contacting a polypeptide with one or more labeled affinity reagents that selectively bind one or more types of terminal amino acids at a terminus of the polypeptide;
ii. identifying a terminal amino acid at the terminus of the polypeptide by detecting an interaction of the polypeptide with the one or more labeled affinity reagents;
iii. removing the terminal amino acid; and
iv. repeating (i)-(iv) one or more times at the terminus of the polypeptide to determine an amino acid sequence of the polypeptide.
274 . The method of claim 273 , wherein the method further comprises:
a. after (i) and before (ii), removing any of the one or more labeled affinity reagents that do not selectively bind the terminal amino acid; and/or
b. after (ii) and before (iii), removing any of the one or more labeled affinity reagents that selectively bind the terminal amino acid.
275 . The method of claim 273 , wherein (iii) comprises modifying the terminal amino acid by contacting the terminal amino acid with an isothiocyanate, and:
a. contacting the modified terminal amino acid with a protease that specifically binds and removes the modified terminal amino acid; or
b. subjecting the modified terminal amino acid to acidic or basic conditions sufficient to remove the modified terminal amino acid.
276 . The method of claim 273 , wherein identifying the terminal amino acid comprises:
a. identifying the terminal amino acid as being one type of the one or more types of terminal amino acids to which the one or more labeled affinity reagents bind; or
b. identifying the terminal amino acid as being a type other than the one or more types of terminal amino acids to which the one or more labeled affinity reagents bind.
277 . The method of claim 273 , wherein the one or more labeled affinity reagents comprise one or more labeled aptamers, one or more labeled peptidases, one or more labeled antibodies, or a combination thereof.
278 . The method of claim 277 , wherein the one or more labeled peptidases have been modified to inactivate cleavage activity; or wherein the one or more labeled peptidases retain cleavage activity for the removing of (iii).
279 . A method of identifying a protein of interest in a mixed sample, the method comprising:
cleaving a mixed protein sample to produce a plurality of polypeptide fragments;
determining an amino acid sequence of at least one polypeptide fragment of the plurality in a method according to claim 273 ; and
identifying a protein of interest in the mixed sample if the amino acid sequence is uniquely identifiable to the protein of interest.
280 . A method of identifying a protein of interest in a mixed sample, the method comprising:
cleaving a mixed protein sample to produce a plurality of polypeptide fragments;
labeling one or more types of amino acids in the plurality of polypeptide fragments with one or more different luminescent labels;
measuring luminescence over time for at least one labeled polypeptide of the plurality;
determining an amino acid sequence of the at least one labeled polypeptide based on the luminescence detected; and
identifying a protein of interest in the mixed sample if the amino acid sequence is uniquely identifiable to the protein of interest.
281 . A method of identifying a relative position of two or more amino acids in a polypeptide, the method comprising:
labeling two or more amino acids of a polypeptide with one or more types of a first FRET label;
detecting a FRET signal from a translocation reaction of the labeled polypeptide in the presence of a cofactor labeled with a second FRET label; and
determining a relative position of the two or more amino acids based on the FRET signal.
282 . A method of preparing a protein for analysis, the method comprising:
i. blocking free carboxylate groups of a protein;
ii. denaturing the protein;
iii. blocking free thiol groups of the protein;
iv. digesting the protein to produce at least one polypeptide fragment comprising a free C-terminal carboxylate group; and
v. chemically conjugating a functional moiety to the free C-terminal carboxylate group.
283 . A method of preparing a protein for analysis, the method comprising:
i. denaturing a protein;
ii. blocking free thiol groups of the protein;
iii. digesting the protein to produce at least one polypeptide fragment comprising a free C-terminal carboxylate group;
iv. blocking the free C-terminal carboxylate group to produce at least one polypeptide fragment comprising a blocked C-terminal carboxylate group; and
v. enzymatically conjugating a functional moiety to the blocked C-terminal carboxylate group.
284 . The method of claim 273 , wherein identifying comprises:
detecting a series of signal pulses indicative of association of the one or more labeled affinity reagents with the polypeptide under polypeptide degradation conditions; and
identifying a first type of amino acid at the terminus of the polypeptide based on a first characteristic pattern in the series of signal pulses.