Proteolytic enzymes play pivotal roles in innumerable physiological and pathological processes; yet the cleavage site specificity of many proteases remains to be determined and numerous putative proteases await functional characterization. Protease specificity relies largely on the active site-mediated recognition of amino acid sequences that encompass the scissile peptide bond, although exosites modify substrate selectivity and enhance catalysis. PICS (Proteome-wide Identification of Protease Cleavage Specificity) is a proteomic technique that employs proteome-derived peptide libraries as proteolytic substrate screens for the characterization of protease active site specificity. To generate the proteome libraries, natural proteomes, such as cell lysates, are endoproteolytically digested into peptides by trypsin or GluC, followed by chemical protection of primary amines. This proteome-derived peptide library is employed as a protease specificity screen following incubation with a test protease. After assay, the neo amino-termini are biotinylated, affinity isolated, and identified by LC-MS/MS. PICS isolates prime-side cleavage products, which, using data base searches, allow to deduce sequence information of the full-length peptidic substrate and so identifies both prime and non-prime sequences and directly determines the cleavage site. The resulting positional information of both prime and nonprime residues also allow investigating subsite cooperativity. With the utilization of proteome derived libraries one can envision using these also for defining the specificity of other PTMs e.g. phosphosites.
Despite its power, PICS does not identify natural substrates from denatured peptides. Global approaches for the identification and analysis of protein terminal peptides are provided by Terminal Amino Isotopic Labelling of Substrates (TAILS, Kleifeld et al Nature Biotech 28, 281-288; Prudova et al 2010 Mol Cell Proteomics) and C-TAILS for the C-terminome (Schilling et al Nature Methods 2010). Our knowledgebase TopFIND (http://clipserve.clip.ubc.ca/topfind) integrates information on N and C-termini, protease cleavage sites and N-terminal modifications and reveals surprisingly widespread modification and truncation of N and C-termini in 4 model species. Notably, we find that one third of the stable proteoforms in a proteome start distal to the expected protein maturation sites such as initiator methionine, signal peptide and pro-peptide removal points. Since the function of a protein can be entirely switched by proteolysis the implications of this are profound for understanding the functional state of a protein, its place in a network and hence the functional state of the proteome.