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.
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Univ Sains Malaysia, Inst Res Mol Med INFORMM, Gelugor 11800, Penang, MalaysiaUniv Sains Malaysia, Inst Res Mol Med INFORMM, Gelugor 11800, Penang, Malaysia
Ujang, Jorim Anak
Kwan, Soon Hong
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Univ Sains Malaysia, Analyt Biochem Res Ctr, Gelugor 11800, Penang, MalaysiaUniv Sains Malaysia, Inst Res Mol Med INFORMM, Gelugor 11800, Penang, Malaysia
Kwan, Soon Hong
Ismail, Mohd Nazri
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Univ Sains Malaysia, Analyt Biochem Res Ctr, Gelugor 11800, Penang, MalaysiaUniv Sains Malaysia, Inst Res Mol Med INFORMM, Gelugor 11800, Penang, Malaysia
Ismail, Mohd Nazri
Lim, Boon Huat
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Univ Sains Malaysia, Sch Hlth Sci, Kubang Kerian 16150, Kelantan, MalaysiaUniv Sains Malaysia, Inst Res Mol Med INFORMM, Gelugor 11800, Penang, Malaysia
Lim, Boon Huat
Noordin, Rahmah
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Univ Sains Malaysia, Inst Res Mol Med INFORMM, Gelugor 11800, Penang, MalaysiaUniv Sains Malaysia, Inst Res Mol Med INFORMM, Gelugor 11800, Penang, Malaysia
Noordin, Rahmah
Othman, Nurulhasanah
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Univ Sains Malaysia, Inst Res Mol Med INFORMM, Gelugor 11800, Penang, MalaysiaUniv Sains Malaysia, Inst Res Mol Med INFORMM, Gelugor 11800, Penang, Malaysia