Authors
Oliver Schilling and Christopher M. Overall
Summary
Specificity of numerous proteases is determined by enzyme-substrate interactions in an extended active site cleft. The Schechter and Berger nomenclature1 designates residues carboxy-terminal to the scissile peptide bond as prime side (P’) and amino-terminal residues as non-prime side (P). P and P’ residues interact with complementary protease subsites called S and S’. While some proteases show very restrictive specificity profiles (e.g. trypsin is specific for lysine and arginine in P1), other proteases display broad specificity profiles involving multiple subsite preferences.
A bottleneck in protease characterization is the determination of consensus cleavage sites as all current techniques provide information on either the prime or nonprime side only−none provide both in the same experiment. Genetic library approaches such as phage display provide cleavable sequences only without identifying the actual scissile bond. Proteome-derived peptide libraries employ biological sequence diversity to robustly characterize protease subsite specificities by mass spectrometry2. Termed Proteomic Identification of protease Cleavage Sites (PICS), peptide libraries are generated from cell lysates or secreted proteins. These libraries are used as substrate screens for test proteases. Prime-side cleavage products are isolated and identified by liquid chromatography/tandem mass spectrometry (LC-MS/MS). The corresponding non-prime side sequences are derived by bioinformatic database searches. Hence, the entire sequence of the peptidic substrate is reconstructed. The cohort of cleaved peptides, often surpassing 200 unique cleavage sequences in single experiments, characterizes subsite preferences (both positive and negative) together with subsite cooperativity. Like any other peptide-based approaches for the determination of active site specificity, PICS does not aim to identify physiological protease substrates.
PICS is optimally suited to determine sequence specificity of endoproteases. Since the method isolates prime-side cleavage products, it is not suited to profile carboxypeptidases as these prime-side cleavage products are too short for LC-MS/MS identification. Many amino peptidases can not cleave at blocked N-termini, which PICS proteome wide libraries feature. PICS utilizes peptide libraries with modified lysine and cysteine residues and hence cannot profile specificity for these residues. PICS screens employing unmodified lysine or cysteine residues are currently under development2.
Proteome-derived peptide libraries are generated by endoproteolytic digestion of proteomes such as cell culture lysates. The specificity of the applied digestion protease is important since it determines sequence features of the peptide library. PICS libraries have been generated with the following three digestion proteases:
− Trypsin cleaves C-terminal to lysine and arginine, hence tryptic peptides lack internal basic residues.
− GluC (Staphylococcus aureus protease V8) cleaves C-terminal to glutamate and, to a lesser extent, aspartate; hence GluC peptides lack internal glutamate residues.
− Chymotrypsin cleaves C-terminal to large hydrophobic residues, hence chymotryptic peptides have a lower internal content of such residues. PICS profiling of matrix metalloprotease (MMP)-2 PICS using chymotryptic libraries confirmed the MMP-2 preference for leucine in P1’, thereby indicating that numerous potential chymotryptic cleavage sites remained intact2.
Profiling proteases using different PICS libraries generated with different digestion proteases provides an unbiased analysis of cleavage site specificity. Our data reported in Schilling and Overall (2008)2 confirms that similar specificity profiles are generated using these different libraries.Proteome-derived peptide libraries were shown to be sufficiently rich in sequence diversity to profile sequence specificity of proteases2. Library generation and isolation of modified peptides can easily be modified to screen the sequence specificity of any posttranslational modification that is amenable to LC-MS/MS detection (e.g phosphorylation or acetylation).
Proteome-derived peptide libraries can also be used to characterize sequence specificity of ligand or receptor binding.
Further details
The protocol was published on Protocol Exchange in 2008. To see the entire protocol, click on the source link.
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