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

J Proteome Res. 2016 Apr 1;15(4):1222-9. doi: 10.1021/acs.jproteome.5b01105. Epub 2016 Mar 22.


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.

Keywords: autolysis protected trypsin; database search; false positives; misassigned spectra; proteomics; search space restriction.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Artifacts*
  • Brain Chemistry
  • Breast Neoplasms / chemistry
  • Cell Line
  • Chromatography, Liquid
  • Data Interpretation, Statistical*
  • Databases, Protein
  • Humans
  • Membrane Proteins / analysis*
  • Membrane Proteins / chemistry
  • Mice
  • Neoplasm Proteins / analysis*
  • Neoplasm Proteins / chemistry
  • Nerve Tissue Proteins / analysis*
  • Nerve Tissue Proteins / chemistry
  • Peptide Fragments / analysis*
  • Protein Structure, Secondary
  • Proteolysis
  • Proteomics / methods
  • Saccharomyces cerevisiae / chemistry
  • Saccharomyces cerevisiae Proteins / analysis*
  • Saccharomyces cerevisiae Proteins / chemistry
  • Tandem Mass Spectrometry
  • Trypsin / chemistry*


  • Membrane Proteins
  • Neoplasm Proteins
  • Nerve Tissue Proteins
  • Numb protein, mouse
  • Peptide Fragments
  • Saccharomyces cerevisiae Proteins
  • Trypsin