PEPTIDE IDENTIFICATION AND SEQUENCING BY SINGLE-MOLECULE DETECTION OF PEPTIDES UNDERGOING DEGRADATION

摘要

The present disclosure provides peptide amino acid sequencing and identification methods and kits for performing such methods. For example, single-molecule detection of fluorophore-labeled peptides is disclosed using multiple rounds of standard Edman degradation or using digestion by chemicals or enzymes. Different fluorophores covalently attached to each of a specific type of amino acid side chain of a peptide provide for the derivation of the peptide's encoded amino acid sequence following image alignments of multiple Edman cycles or following digestion by chemicals or enzymes. The amino acid sequence of a peptide and/or the identity of the peptide can be determined by bioinformatic analysis based on the encoded amino acid sequence. The present disclosure further provides peptide derivatization and immobilization strategies to enable the sequencing and identification of a single peptide or a plurality of peptides.

主权项

1. A method for sequencing one or more than one peptide comprising:
a. labeling the amino acid side chain of one or more amino acid of a first type with a first detectable moiety, wherein said first detectable moiety selectively labels the side chain characterizing said one or more amino acid of a first type; b. labeling the amino acid side chain of one or more amino acid of a second type with a second detectable moiety, wherein said second detectable moiety selectively labels the side chain characterizing said one or more amino acid of a second type; c. attaching said peptide to a surface; d. imaging said peptide; e. cleaving said peptide; f. imaging said peptide after the cleavage of step (e); g. repeating steps (e) to (f) as necessary; h. comparing the image of step (d) with the image of step (f) and identifying a change or an absence of a change in the image between step (d) and step (f); i. if further cleavage is performed as in step (g), comparing the image before and after each subsequent cleavage step (e) and identifying a change or an absence of a change in the image; and j. determining the sequence of the peptide based on at least one change or at least one absence of a change in the image identified in step (h) or (i).