title: Characterization of the prime and non-prime active site specificities of proteases by proteome-derived peptide libraries and tandem mass spectrometry.
abstract: 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.
authors.affiliation: Institute for Molecular Medicine and Cell Research, University of Freiburg, Freiburg, Germany.
authors.forename: Oliver
authors.initials: O
authors.lastname: Schilling
authors.forename: Pitter F
authors.initials: PF
authors.lastname: Huesgen
authors.forename: Olivier
authors.initials: O
authors.lastname: Barré
authors.forename: Ulrich
authors.initials: U
authors.lastname: Auf dem Keller
authors.forename: Christopher M
authors.initials: CM
authors.lastname: Overall
mesh_terms: D020134:Catalytic Domain; D002853:Chromatography, Liquid / Q000379:methods; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D020407:Internet; D010447:Peptide Hydrolases / Q000737:chemistry*; D019151:Peptide Library; D020543:Proteome; D020539:Sequence Analysis, Protein; D012984:Software; D013379:Substrate Specificity; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D019151:Peptide Library; D020543:Proteome; D010447:Peptide Hydrolases