title: Cleaning out the Litterbox of Proteomic Scientists' Favorite Pet: Optimized Data Analysis Avoiding Trypsin Artifacts.
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.
authors.affiliation: Research Unit Functional Proteomics and Metabolic Pathways, Institute of Pathology, Medical University of Graz , 8010 Graz, Austria.|Omics Center Graz, BioTechMed-Graz , 8010 Graz, Austria.
authors.forename: Matthias
authors.initials: M
authors.lastname: Schittmayer
authors.affiliation: Research Unit Functional Proteomics and Metabolic Pathways, Institute of Pathology, Medical University of Graz , 8010 Graz, Austria.|Omics Center Graz, BioTechMed-Graz , 8010 Graz, Austria.
authors.forename: Katarina
authors.initials: K
authors.lastname: Fritz
authors.affiliation: Research Unit Functional Proteomics and Metabolic Pathways, Institute of Pathology, Medical University of Graz , 8010 Graz, Austria.|Omics Center Graz, BioTechMed-Graz , 8010 Graz, Austria.
authors.forename: Laura
authors.initials: L
authors.lastname: Liesinger
authors.affiliation: Department of Dermatology, Medical University of Vienna , 1090 Vienna, Austria.
authors.forename: Johannes
authors.initials: J
authors.lastname: Griss
authors.affiliation: Research Unit Functional Proteomics and Metabolic Pathways, Institute of Pathology, Medical University of Graz , 8010 Graz, Austria.|Omics Center Graz, BioTechMed-Graz , 8010 Graz, Austria.
authors.forename: Ruth
authors.initials: R
authors.lastname: Birner-Gruenberger
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D016477:Artifacts; D001923:Brain Chemistry; D001943:Breast Neoplasms / Q000737:chemistry; D002460:Cell Line; D002853:Chromatography, Liquid; D003627:Data Interpretation, Statistical; D030562:Databases, Protein; D006801:Humans; D008565:Membrane Proteins / Q000032:analysis* / Q000737:chemistry; D051379:Mice; D009363:Neoplasm Proteins / Q000032:analysis* / Q000737:chemistry; D009419:Nerve Tissue Proteins / Q000032:analysis* / Q000737:chemistry; D010446:Peptide Fragments / Q000032:analysis*; D017433:Protein Structure, Secondary; D059748:Proteolysis; D040901:Proteomics / Q000379:methods; D012441:Saccharomyces cerevisiae / Q000737:chemistry; D029701:Saccharomyces cerevisiae Proteins / Q000032:analysis* / Q000737:chemistry; D053719:Tandem Mass Spectrometry; D014357:Trypsin / Q000737:chemistry*
chemical_list: D008565:Membrane Proteins; D009363:Neoplasm Proteins; D009419:Nerve Tissue Proteins; C106293:Numb protein, mouse; D010446:Peptide Fragments; D029701:Saccharomyces cerevisiae Proteins; D014357:Trypsin
keywords: autolysis protected trypsin; database search; false positives; misassigned spectra; proteomics; search space restriction