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Protease specificity profiling by tandem mass spectrometry using proteome-derived peptide libraries.
Oliver Schilling1, Ulrich auf dem Keller, Christopher M Overall
1Institute for Molecular Medicine and Cell Research, University of Freiburg, Freiburg, Germany. oliver.schilling@mol-med.unifreiburg.de
Methods in Molecular Biology (Clifton, N.J.)
|May 24, 2011
Summary
Protease specificity profiling using Proteomic Identification of protease Cleavage Sites (PICS) offers a novel method to define enzyme active sites. This approach uses proteome-derived peptide libraries for comprehensive protease specificity analysis.
Area of Science:
- Biochemistry
- Proteomics
- Enzymology
Background:
- Protease specificity is crucial for biological processes.
- Existing methods for protease specificity profiling have limitations.
Purpose of the Study:
- To introduce and validate a novel method, PICS, for comprehensive protease specificity profiling.
- To describe a simplified procedure for PICS peptide library generation and assay performance.
Main Methods:
- Generation of proteome-derived peptide libraries by digestion with specific proteases (trypsin, GluC, chymotrypsin).
- Chemical protection of primary amines followed by biotinylation of neo-N-termini after test protease incubation.
- Enrichment and identification of peptides using liquid chromatography-tandem mass spectrometry (LC-MS/MS).
- Bioinformatic derivation of nonprime-side sequences.
Main Results:
- PICS simultaneously profiles both prime and nonprime protease specificities.
- Direct determination of hundreds of cleavage site sequences in a single experiment.
- Enables investigation of subsite cooperativity.
Conclusions:
- PICS provides a powerful and efficient approach for detailed protease specificity profiling.
- The method is applicable to most protease classes, excluding specific types requiring free alpha-amines.
- PICS generates extensive sequence specificity data for a deeper understanding of protease function.
