Figure 3 -. Peptide-level analysis of protein targets.

a) Cysteines on the protein of interest are reduced and alkylated, followed by digestion of the protein target. b) During MS/MS peptides tend to dissociate at amide bonds, generating a ‘ladder’ of fragment ions from which the amino acid sequence is derived. Accepted nomenclature refers to fragments containing the original peptide c-terminus as ‘y-type’ and fragments with the original n-terminus as ‘b-type’. In this example, red glyphs immediately below the peptide indicate amino acids identified by fragment ions (red) in the MS/MS spectrum. c) Peptide-level analysis of proteins provides information about the percentage occupancy of the inhibitor and identifies site of labelling. Inhibitor-specific ion, used to improve peptide sequence identification, is labelled by green *. d) Peptide-level analysis of proteins provides information about specificity of inhibitor labelling, even when the apparent binding stoichiometry is 1:1.