Peptide identification and sequencing by single-molecule detection of peptides undergoing degradation

A method for identifying a 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 by chemical or enzymatic digestion;
• 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 any 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 any change in the image;
• j. comparing at least one change in the image identified in step (h) or (i) to a database of changes in the images of known protein sequences due to equivalent cleavage;
• and
• k. identifying the peptide.

A method for sequencing a peptide and determining the presence or absence of a post-translational modification of said 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. labeling said peptide such that a post-translational modification, if present, is labeled in a manner distinct from the labeling of any amino acid side chain;
• d. attaching said peptide to a surface;
• e. imaging said peptide;
• f. cleaving said peptide;
• g. imaging said peptide after the cleavage of step (f);
• h. repeating steps (f) to (g) as necessary;
• i. comparing the image of step (e) with the image of step (g) and identifying a change or an absence of a change in the image between step (e) and step (g);
• j. if further cleavage is performed as in step (h), comparing the image before and after each subsequent cleavage step (f) and identifying a change or an absence of a change in the image;
• k. comparing at least one change or at least one absence of a change in the image identified in step (i) or (j) to a database of image information of known protein sequences;
• l. determining the sequence of the peptide based on the comparison of step (k);
• and
• m. determining the presence or absence of a post-translational modification of said peptide based on the imaging of step (e) of the labeling of a post-translation modification, if present, of step (c).