Cleaning out the Litterbox of Proteomic Scientists’ Favorite Pet: Optimized Data Analysis Avoiding Trypsin Artifacts

Chemically
modified trypsin is a standard reagent in proteomics
experiments but is usually not considered in database searches. Modification
of trypsin is supposed to protect the protease against autolysis and
the resulting loss of activity. Here, we show that modified trypsin
is still subject to self-digestion, and, as a result, modified trypsin-derived
peptides are present in standard digests. We depict that these peptides
commonly lead to false-positive assignments even if native trypsin
is considered in the database. Moreover, we present an easily implementable
method to include modified trypsin in the database search with a minimal
increase in search time and search space while efficiently avoiding
these false-positive hits.