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Poster Presentations
Day 3, June 24（Tue.）
Room P (Maesato East, Foyer, Ocean Wing)
- 3P-PM-16（2C-O2-1510）
Advancing Bottom-up Proteomics with Protease Type XIII from Aspergillus saitoi
(1Kyoto Univ., 2SHIONOGI, 3NIBN)
oRyota Tomioka1,2, Ayana Tomioka1, Kosuke Ogata1, Yasushi Ishihama1,3
Bottom-up proteomics is a powerful technique for comprehensive analysis of proteins by cleaving proteins into peptides with proteases and analyzing them by liquid chromatography/tandem mass spectrometry. Trypsin is the gold standard protease for bottom-up proteomics however its cleavage specificity limits peptide identification depending on the protein sequences. In addition, its optimal pH conditions are weakly alkaline, which can cause modification artifacts such as deamidation. To address these limitations, we investigated protease type XIII (P13ase) from Aspergillus saitoi as a non-specific protease that is active at low pH. Recently, P13ase has been used in hydrogen deuterium exchange mass spectrometry for protein structural analysis, however its properties such as cleavage preferences have not been fully characterized. In this study, we examined the optimal digestion conditions for P13ase and found that digestion at pH 3.5, 37 °C for 60 minutes was optimal for bottom-up proteomics. The optimized digestion conditions improved sequence coverage of some proteins compared to trypsin. In addition, the P13ase digestion reduced artifacts including deamidation and cyclization of Asn and Gln. These results support P13ase as a valuable tool for bottom-up proteomics, and the use of P13ase is expected to enable more precise proteomics studies.