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

Abstract

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. Molecular & Cellular

Infection of HeLa cells with Chlamydia trachomatis inhibits protein synthesis and causes multiple changes to host cell pathways

“…Liquid chromatography–tandem mass spectrometry measurements using a Q‐Exactive+ were performed as described (Biniossek et al, ). MS data were analysed by MaxQuant (Cox & Mann, ) version 1.5.28 with the Uniprot human database downloaded on November 2015, counting 20,193 reviewed entries.…”

“…Liquid chromatography-tandem mass spectrometry measurements using a Q-Exactive+ were performed as described (Biniossek et al, 2016). MS data were analysed by MaxQuant (Cox & Mann, 2008…”

Global substrate specificity profiling of post‐translational modifying enzymes

“…Mass spectrometry in combination with proteome‐derived substrate libraries has been successfully applied to define protease specificity . These “degradomics” methods use liquid chromatography with tandem mass spectrometry (LC‐MS/MS) to identify the site of proteolytic cleavage within proteome‐derived peptides and can define prime side and nonprime side specificity determinants in a single assay.…”

“…Mass spectrometry in combination with proteomederived substrate libraries has been successfully applied to define protease specificity. [56][57][58][59][60][61] These "degradomics" methods use liquid chromatography with tandem mass spectrometry (LC-MS/MS) to identify the site of proteolytic cleavage within proteome-derived peptides and can define prime side and nonprime side specificity determinants in a single assay. These techniques are quite powerful, but require chemical labeling steps for enrichment and identification of neo-termini and present a challenge in extracting kinetic parameters.…”

Monitoring proteolytic processing events by quantitative mass spectrometry

“…By contrast, threonine, cysteine, and serine proteases catalyze peptide cleavage using an amino acid in the binding pocket (either Thr, Cys, or Ser) as a nucleophile [2] For a more in-depth description of protease families, please refer to [7]. Although this process is highly regulated, aberrant protease activity is a hallmark for several diseases including neurodegeneration, autoimmune disorders, and cancer [2, 8]. Serine proteases for example, are overexpressed in certain types of colorectal cancer.…”