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

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

Author information

1
Research Unit Functional Proteomics and Metabolic Pathways, Institute of Pathology, Medical University of Graz , 8010 Graz, Austria.
2
Omics Center Graz, BioTechMed-Graz , 8010 Graz, Austria.
3
Department of Dermatology, Medical University of Vienna , 1090 Vienna, Austria.

Abstract

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

PMID:
26938934
PMCID:
PMC4820788
DOI:
10.1021/acs.jproteome.5b01105
[Indexed for MEDLINE]
Free PMC Article

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