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 se...

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Bibliographic Details
Published inJournal of proteome research Vol. 15; no. 4; pp. 1222 - 1229
Main Authors Schittmayer, Matthias, Fritz, Katarina, Liesinger, Laura, Griss, Johannes, Birner-Gruenberger, Ruth
Format Journal Article
LanguageEnglish
Published United States American Chemical Society 01.04.2016
Subjects
Amino Acid Sequence
Animals
autolysis
Brain Chemistry
Breast Neoplasms - chemistry
Cell Line
Chromatography, Liquid
data analysis
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
peptides
Protein Structure, Secondary
Proteolysis
proteome
proteomics
Proteomics - methods
Saccharomyces cerevisiae - chemistry
Saccharomyces cerevisiae Proteins - analysis
Saccharomyces cerevisiae Proteins - chemistry
Tandem Mass Spectrometry
trypsin
Trypsin - chemistry
autolysis protected trypsin
database search
misassigned spectra
search space restriction
proteomics
false positives
Online AccessGet full text
ISSN1535-3893
1535-3907
1535-3907
DOI10.1021/acs.jproteome.5b01105

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Summary: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.
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ISSN:1535-3893
1535-3907
1535-3907
DOI:10.1021/acs.jproteome.5b01105