Proteomics

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Tag-Free Identification of Protease Specificity Using Proteome-Derived Peptide Libraries


ABSTRACT: Proteome-derived peptide libraries are a powerful tool for the investigation of protease specificity, conventionally involving the biotinylation of cleavage products to enable for their enrichment. Here we present a modified strategy for protease specificity profiling using proteome-derived peptide libraries in a tag-free manner. In comparison to the original method the new protocol is faster, avoids cost- and time-intensive enrichment steps, and enables probing for lysine selectivity. In the new workflow, peptide libraries are generated by endoproteolytic digestion of proteomes without chemical modification of primary amines prior to exposure to a protease under investigation. After incubation with the test protease, treated and control library are differentially labeled using stable isotopes by means of reductive dimethylation. Upon analysis by liquid chromatography mass spectrometry, cleavage products of the test protease appear as peptides enriched for the respective isotopic label. With “PICS 2.0” we identified more than 2800 cleavage sites in five specificity experiments for four proteases, including trypsin, caspase-3, and chlamydial protease-like activity factor (CPAF). Hence, this method is a valuable addition to the different strategies currently employed for specificity profiling of proteases

INSTRUMENT(S): Q Exactive

ORGANISM(S): Escherichia Coli

SUBMITTER: Oliver Schilling  

LAB HEAD: Oliver Schilling

PROVIDER: PXD002785 | Pride | 2016-05-02

REPOSITORIES: Pride

Dataset's files

Source:
Action DRS
ATS15-LysC-BM2689.raw Raw
ATS15-LysC.interact.pep.xml Pepxml
ATS15-LysC.mzXML Mzxml
ATS15-trypsin-BM2672.raw Raw
ATS15-tryptic.interact.pep.xml Pepxml
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Publications

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

Biniossek Martin L ML   Niemer Melanie M   Maksimchuk Ken K   Mayer Bettina B   Fuchs Julian J   Huesgen Pitter F PF   McCafferty Dewey G DG   Turk Boris B   Fritz Guenther G   Mayer Jens J   Haecker Georg G   Mach Lukas L   Schilling Oliver O  

Molecular & cellular proteomics : MCP 20160427 7


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  ...[more]

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