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.
Highlights
After inactivation of the digestion protease, the peptide library is divided into a control sample and a sample for incubation with a protease under investigation
The requirement that the semi-specific peptide needs to be present only in the test protease treated sample but not in the control excludes artifacts arising from unspecific background proteolysis in the proteome
Using the human endogenous retroviruses (HERVs)-K(HML-2) dataset, we investigated whether the appearance of cleavage products coincides with the depletion of the original tryptic peptides
Summary
C-terminal, carboxy-terminal; cDNA, complementary DNA; CPAF, chlamydial protease-like activity factor; HERV, human endogenous retrovirus; LC–MS/MS, liquid chromatography–tandem mass spectrometry; N-terminal, aminoterminal; P, nonprime side; P’, prime side; PICS, proteomic identification of protease cleavage sites. Synthetic mixture-based oriented peptide libraries represent a two-step strategy to characterize proteases with specificity profiles that include both prime and nonprime sites (7). Proteome-derived peptide libraries have been introduced for the specificity profiling of proteases (5), including cysteine, serine, and metalloproteases (5, 20, 21) with adaptions to enable multiplexed stable isotope tagging for kinetic investigations (22) as well as investigating the specificity of carboxypeptidases and N␣-acetyltransferases (23, 24). 3502 of the 3988 (88%) proteases annotated in the MEROPS database (version 9.12) (26) have less than 10 known substrates This highlights that deorphanizing remains an important goal in protease research and underlines the need for straightforward approaches for protease cleavage site identification
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