10.1021/pr4001465.s003 Jakob Bunkenborg Jakob Bunkenborg Guadalupe Espadas Guadalupe Espadas Henrik Molina Henrik Molina Cutting Edge Proteomics: Benchmarking of Six Commercial Trypsins American Chemical Society 2013 TMT species Commercial TrypsinsTryptic digestion LC tandem mass tag peptide autolysis products trypsin 2013-08-02 00:00:00 Journal contribution https://acs.figshare.com/articles/journal_contribution/Cutting_Edge_Proteomics_Benchmarking_of_Six_Commercial_Trypsins/2391898 Tryptic digestion is an important component of most proteomics experiments, and trypsin is available from many sources with a cost that varies by more than 1000-fold. This high-mass-accuracy LC–MS study benchmarks six commercially available trypsins with respect to autolytic species and sequence specificity. The analysis of autolysis products led to the identification of a number of contaminating proteins and the generation of a list of peptide species that will be present in tryptic digests. Intriguingly, many of the autolysis products were nontryptic peptides, specifically peptides generated by C-terminal cleavage at asparagine residues. Both porcine and bovine trypsins were demonstrated to be tyrosine O-sulfated. Using both a label-free and a tandem mass tag (TMT) labeling approach, a comparison of the digestion of a standard protein mixture using the six trypsins demonstrated that, apart from the least expensive bovine trypsin, the trypsins were equally specific. The semitryptic activity led to a better sequence coverage for abundant substrates at the expense of low-abundance species. The label-free analysis was shown to be more sensitive to unique features from the individual digests that were lost in the TMT-multiplexing study.