Identification of Protease Specificity by Combining Proteome-Derived Peptide Libraries and Quantitative Proteomics.

Journal Article (Journal Article)

We present protease specificity profiling based on quantitative proteomics in combination with proteome-derived peptide libraries. Peptide libraries are generated by endoproteolytic digestion of proteomes without chemical modification of primary amines before exposure to a protease under investigation. After incubation with a test protease, treated and control libraries are differentially isotope-labeled using cost-effective reductive dimethylation. Upon analysis by liquid chromatography-tandem mass spectrometry, cleavage products of the test protease appear as semi-specific peptides that are enriched for the corresponding isotope label. We validate our workflow with two proteases with well-characterized specificity profiles: trypsin and caspase-3. We provide the first specificity profile of a protease encoded by a human endogenous retrovirus and for chlamydial protease-like activity factor (CPAF). For CPAF, we also highlight the structural basis of negative subsite cooperativity between subsites S1 and S2'. For A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) -4, -5, and -15, we show a canonical preference profile, including glutamate in P1 and glycine in P3'. In total, we report nearly 4000 cleavage sites for seven proteases. Our protocol is fast, avoids enrichment or synthesis steps, and enables probing for lysine selectivity as well as subsite cooperativity. Due to its simplicity, we anticipate usability by most proteomic laboratories.

Full Text

Duke Authors

Cited Authors

  • Biniossek, ML; Niemer, M; Maksimchuk, K; Mayer, B; Fuchs, J; Huesgen, PF; McCafferty, DG; Turk, B; Fritz, G; Mayer, J; Haecker, G; Mach, L; Schilling, O

Published Date

  • July 2016

Published In

Volume / Issue

  • 15 / 7

Start / End Page

  • 2515 - 2524

PubMed ID

  • 27122596

Pubmed Central ID

  • PMC4937521

Electronic International Standard Serial Number (EISSN)

  • 1535-9484

International Standard Serial Number (ISSN)

  • 1535-9476

Digital Object Identifier (DOI)

  • 10.1074/mcp.o115.056671


  • eng