Proteomics

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All under acidic bottom-up proteomics using protease type XIII from Aspergillus Saitoi

ABSTRACT: Bottom-up proteomics is a powerful technique for comprehensive analysis of proteins by cleaving them into peptides with proteases and analysing 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 sequence. In addition, its optimal pH conditions are weakly alkaline, which can cause modification artifacts such as deamidation. In addressing these limitations, we have investigated the potential of protease type XIII (P13ase) from Aspergillus saitoi, which is active at low pH. P13ase has been used for protein structural analysis by hydrogen deuterium exchange mass spectrometry, but its cleavage preferences has not been clarified. In this study, we found that the optimal P13ase digestion conditions for bottom-up proteomics are pH 3.5, 37°C for 60 min, and that P13ase primarily cleaves the C-terminal side of Lys, Arg, and Leu. When this optimal digestion condition was applied to HeLa cell extracts and compared to trypsin, it was possible to achieve sequence coverage of more than 90% for several proteins, which is unachievable with trypsin. In addition, P13ase digestion reduced artifacts such as deamidation products caused by cyclization reactions and subsequent hydrolysis. The results of this study support the high potential of P13ase as a new tool for precision proteomics.

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Yasushi Ishihama 

PROVIDER: PXD064095 | JPOST Repository | Fri Nov 28 00:00:00 GMT 2025

REPOSITORIES: jPOST

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240602RT_trypsin_1.raw Raw
240602RT_trypsin_2.raw Raw
240602RT_trypsin_3.raw Raw
240602RT_typeXIII_pH1p5_1.raw Raw
240602RT_typeXIII_pH1p5_2.raw Raw
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Bottom-up proteomics under acidic conditions using protease type XIII from Aspergillus saitoi.

Tomioka Ryota R   Tomioka Ayana A   Ogata Kosuke K   Ishihama Yasushi Y  

Molecular & cellular proteomics : MCP 20251115

Bottom-up proteomics is a powerful technique for comprehensive analysis of proteins by proteolytic cleavage followed by liquid chromatography/tandem mass spectrometry to identify the resulting peptides. Trypsin is the gold-standard protease for bottom-up proteomics, though its cleavage specificity limits peptide identification, depending on the protein sequence. In addition, its optimal pH is weakly alkaline, which can cause modification artifacts such as deamidation. We hypothesized that these  ...[more]

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