title: Quantitative protease cleavage site profiling using tandem-mass-tag labeling and LC-MALDI-TOF/TOF MS/MS analysis.
abstract: Knowledge of cleavage site specificity and activity are major prerequisites for understanding protease function. On the basis of a recently presented approach for proteomic identification of cleavage sites (PICS) in proteome-derived peptide libraries, we developed an isobaric labeling quantitative LC-MALDI-TOF/TOF MS/MS approach (Q-PICS) for simultaneous determination of cleavage site specificity and robust relative quantification of proteolytic events. For GluC-protease, 737 cleavage sites were identified in a yeast proteome-derived peptide library; 94.0% showed the typical GluC specificity for peptide bonds at glutamyl and aspartyl residues. The six-plex tandem mass tagging strategy allowed for three simultaneous replicates in a single run, guaranteeing high confidence and robust statistics for quantitative measurements. Using the quantitative capacity of Q-PICS, we performed a comparison of cleavage site specificity of GluC in two different buffer systems. The results support earlier findings describing that apparent difference between the buffer systems are probably caused by the inhibitory effect of bicarbonate on the overall GluC activity and that the preference for Glu-X bonds compared to Asp-X bonds is independent of the buffer system used.
authors.affiliation: AG Systematische Proteomforschung, Institut für Experimentelle Medizin, Christian-Albrechts-Universität zu Kiel, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Jakoby
authors.forename: Bart Hj
authors.initials: BH
authors.lastname: van den Berg
authors.forename: Andreas
authors.initials: A
authors.lastname: Tholey
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D002021:Buffers; D002853:Chromatography, Liquid; D010450:Endopeptidases / Q000737:chemistry*; D057075:Enzyme Assays; D010446:Peptide Fragments / Q000737:chemistry; D010449:Peptide Mapping; D059748:Proteolysis; D020543:Proteome / Q000737:chemistry; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry; D020539:Sequence Analysis, Protein / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013194:Staining and Labeling / Q000379:methods*; D013379:Substrate Specificity; D053719:Tandem Mass Spectrometry
chemical_list: D002021:Buffers; D010446:Peptide Fragments; D020543:Proteome; D029701:Saccharomyces cerevisiae Proteins; D010450:Endopeptidases