Maximizing Cumulative Trypsin Activity with Calcium at Elevated Temperature for Enhanced Bottom-Up Proteome Analysis.

ABSTRACT: Bottom-up proteomics relies on efficient trypsin digestion ahead of MS analysis. Prior studies have suggested digestion at elevated temperature to accelerate proteolysis, showing an increase in the number of MS-identified peptides. However, improved sequence coverage may be a consequence of partial digestion, as higher temperatures destabilize and degrade the enzyme, causing enhanced activity to be short-lived. Here, we use a spectroscopic (BAEE) assay to quantify calcium-stabilized trypsin activity over the complete time course of a digestion. At 47 °C, the addition of calcium contributes a 25-fold enhancement in trypsin stability. Higher temperatures show a net decrease in cumulative trypsin activity. Through bottom-up MS analysis of a yeast proteome extract, we demonstrate that a 1 h di

SUBMITTER: Nickerson JL 

PROVIDER: S-EPMC9598648 | biostudies-literature | 2022 Oct

REPOSITORIES: biostudies-literature