Non-specific Digestion Artifacts of Bovine Trypsin Exemplified with Surrogate Peptides for Endogenous Protein Quantitation

Abstract

In bottom-up MS-based proteomic workflows, peptides are used as surrogates of proteins either for identification or quantification. Trypsin is the most preferred and widely used protease to generate these surrogate peptides for peptide based quantitative targeted proteomics. During one of such surrogate based protein quantitations, one of the experiments to understand the stability of the selected surrogate peptides during the process of digestion, yielded very low recovery (2%) compared to an equimolar amount of the pure peptide. This prompted us to investigate the reasons using high-resolution mass spectrometry (Q-Exactive plus) with data dependent MS2 acquisition (ddMS2) and parallel reaction monitoring (PRM) experiments. Investigations revealed non-specific cleavage of the peptide bond predominantly after phenylalanine (F) and tyrosine (Y) amino acid residues of the surrogate peptide standard by trypsin. PRM data showed high abundance of these truncated peptides compared to full length peptide, thus explaining the low recovery of peptide standards during the digestion process. Further investigations suggested a possible role of pseudo-trypsin (with chymotrypsin like specificity), which is likely formed from autolysis of trypsin during the course of digestion. Sequence grade trypsin (TPCK treated and reductively methylated) did not show any such non-specific cleavage of the peptides. Our findings, therefore, demonstrate the importance of choosing the appropriate trypsin for protein quantitation experiments. The propensity of trypsin that can form pseudotrypsin can produce non-specific digestion artifacts that can under predict the protein concentrations in surrogate peptide based quantification methods.