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

## TL;DR

A new acidic protease from Aspergillus saitoi improves bottom-up proteomics by reducing artifacts and increasing sequence coverage.

## Contribution

P13ase's cleavage preferences and acidic digestion conditions are characterized for precision proteomics.

## Key findings

- P13ase cleaves preferentially at leucine, lysine, and arginine residues.
- P13ase achieves over 90% sequence coverage at pH 3.5, outperforming trypsin.
- P13ase reduces deamidation artifacts due to its acidic optimal pH.

## Abstract

Bottom-up proteomics is a powerful technique for comprehensive analysis of proteins by proteolytic cleavage followed by LC-MS/MS 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 limitations might be overcome by using 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 have not been clarified. Here, we demonstrated that P13ase exhibits a preference for cleaving the C-terminal sides of leucine (21%), lysine (19%), and arginine (15%) residues, and that the optimal conditions for P13ase digestion in bottom-up proteomics are pH 3.5 and 37 °C for 60 min. Under these conditions, sequence coverage of more than 90% was achieved for several proteins in HeLa cell extracts, which is unachievable with trypsin. In addition, P13ase digestion reduced artifacts such as deamidation products generated by cyclization reactions and subsequent hydrolysis. These results indicate that P13ase is a promising new tool for precision proteomics.

•The cleavage preferences of P13ase were characterized.•P13ase minimizes artifacts such as deamidation because of its acidic optimal pH.•P13ase shows comparable proteome depth and quantification to trypsin.

The cleavage preferences of P13ase were characterized.

P13ase minimizes artifacts such as deamidation because of its acidic optimal pH.

P13ase shows comparable proteome depth and quantification to trypsin.

This study evaluates the suitability of protease type XIII (P13ase) from Aspergillus saitoi for bottom-up proteomics, focusing on optimal digestion conditions, cleavage preferences, and artifact minimization. P13ase shows unique cleavage patterns affording high sequence coverage, and its acidic optimal pH reduces deamidation artifacts compared with trypsin.