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Bottom-Up Proteomics Under Acidic Conditions Using Protease Type XIII From Aspergillus saitoi.

Ryota Tomioka1, Ayana Tomioka2, Kosuke Ogata2

  • 1Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto, Japan; Shionogi & Co, Ltd, Biopharmaceutical Research Division, Toyonaka, Osaka, Japan.

Molecular & Cellular Proteomics : MCP
|November 17, 2025
PubMed
Summary

Protease type XIII (P13ase) offers improved protein sequence coverage in bottom-up proteomics compared to trypsin. This novel protease functions optimally at low pH, reducing common artifacts and enhancing precision proteomics.

Keywords:
bottom-up proteomicscleavage preferencedeamidationproteaseprotease type XIII

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Area of Science:

  • Proteomics
  • Mass Spectrometry
  • Enzymology

Background:

  • Bottom-up proteomics relies on proteolytic cleavage for peptide identification.
  • Trypsin, the standard protease, has limitations including cleavage specificity and artifact generation at alkaline pH.

Purpose of the Study:

  • To evaluate Protease type XIII (P13ase) from Aspergillus saitoi as an alternative protease for bottom-up proteomics.
  • To characterize P13ase cleavage preferences and optimal digestion conditions.
  • To assess P13ase's ability to improve sequence coverage and reduce artifacts compared to trypsin.

Main Methods:

  • Proteolytic digestion of proteins using P13ase under varying conditions.
  • Liquid chromatography-tandem mass spectrometry (LC-MS/MS) for peptide identification.
  • Comparison of P13ase digestion with trypsin digestion using HeLa cell extracts.

Main Results:

  • P13ase demonstrated cleavage preference for leucine, lysine, and arginine residues.
  • Optimal P13ase digestion conditions were identified as pH 3.5 and 37 °C for 60 minutes.
  • Over 90% protein sequence coverage was achieved with P13ase, exceeding trypsin's performance.
  • P13ase digestion significantly reduced deamidation artifacts.

Conclusions:

  • P13ase is a promising protease for bottom-up proteomics, offering enhanced sequence coverage.
  • Low-pH activity of P13ase minimizes artifact formation, leading to more accurate protein analysis.
  • P13ase represents a valuable new tool for precision proteomics.