Cutting Edge Proteomics: Benchmarking of Six Commercial Trypsins

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.