Global identification of peptidase specificity by multiplex substrate profilingO'Donoghue AJ, Eroy-Reveles AA, Knudsen GM, Ingram J, Zhou M, Statnekov JB, Greninger AL, Hostetter DR, Qu G, Maltby DA, Anderson MO, Derisi JL, McKerrow JH, Burlingame AL, Craik CS.
Nat Methods, 2012Abstract: We developed a simple and rapid multiplex substrate-profiling method to reveal
the substrate specificity of any endo- or exopeptidase using liquid
chromatography-tandem mass spectrometry sequencing. We generated a
physicochemically diverse library of peptides by incorporating all combinations
of neighbor and near-neighbor amino acid pairs into decapeptide sequences that
are flanked by unique dipeptides at each terminus. Addition of a panel of
evolutionarily diverse peptidases to a mixture of these tetradecapeptides
generated information on prime and nonprime sites as well as on substrate
specificity that matched or expanded upon known substrate motifs. This method
biochemically confirmed the activity of the klassevirus 3C protein responsible
for polypeptide processing and allowed granzyme B substrates to be ranked by
enzymatic turnover efficiency using label-free quantitation of precursor-ion
abundance. Additionally, the proteolytic secretions from schistosome parasitic
flatworm larvae and a pancreatic cancer cell line were deconvoluted in a
subtractive strategy using class-specific peptidase inhibitors
