Journal
NATURE PROTOCOLS
Volume 6, Issue 1, Pages 111-120Publisher
NATURE PUBLISHING GROUP
DOI: 10.1038/nprot.2010.178
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Funding
- Deutsche Forschungsgemeinschaft (DFG) [SCHI 871/1-1, 871/2-1]
- Michael Smith Foundation for Health Research (MSFHR)
- German Academic Exchange Service (DAAD)
- MSFHR
- Swiss National Foundation of Sciences (SNF)
- Canadian Institutes of Health Research (CIHR)
- Canada Research Chair in Metalloproteinase Proteomics and Systems Biology
- CIHR
- Canadian Breast Cancer Research Alliance (CBCRA)
- Canadian Breast Cancer Foundation
- Cancer Research Society
- MSHFR
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To link cleaved substrates in complex systems with a specific protease, the protease active site specificity is required. Proteomic identification of cleavage sites (PICS) simultaneously determines both the prime-and non-prime-side specificities of individual proteases through identification of hundreds of individual cleavage sequences from biologically relevant, proteome-derived peptide libraries. PICS also identifies subsite cooperativity. To generate PICS peptide libraries, cellular proteomes are digested with a specific protease such as trypsin. Following protease inactivation, primary amines are protected. After incubation with a test protease, each prime-side cleavage fragment has a free newly formed N-terminus, which is biotinylated for affinity isolation and identification by liquid chromatography-tandem mass spectrometry. The corresponding non-prime sequences are derived bioinformatically. The step-by-step protocol also presents a web service for PICS data analysis, as well as introducing and validating PICS peptide libraries made from Escherichia coli.
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