Global identification of peptidase specificity by multiplex substrate profiling

Nat Methods. 2012 Nov;9(11):1095-100. doi: 10.1038/nmeth.2182. Epub 2012 Sep 30.


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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Validation Study

MeSH terms

  • 3C Viral Proteases
  • Animals
  • Carboxypeptidases / metabolism
  • Carcinoma, Pancreatic Ductal / enzymology
  • Cathepsin E / metabolism
  • Cell Line, Tumor
  • Chromatography, Liquid
  • Cysteine Endopeptidases / metabolism
  • Exopeptidases / metabolism
  • Granzymes / metabolism
  • Humans
  • Mice
  • Pancreatic Elastase / metabolism
  • Peptide Hydrolases / metabolism*
  • Peptide Library
  • Peptides / metabolism
  • Schistosoma mansoni
  • Substrate Specificity*
  • Tandem Mass Spectrometry
  • Viral Proteins / metabolism


  • Peptide Library
  • Peptides
  • Viral Proteins
  • Carboxypeptidases
  • Exopeptidases
  • Peptide Hydrolases
  • lysosomal Pro-X carboxypeptidase
  • Granzymes
  • Pancreatic Elastase
  • Cysteine Endopeptidases
  • 3C Viral Proteases
  • 3C proteases
  • Cathepsin E