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Systematic determination of ion score cutoffs based on calculated false positive rates: application for identifying ubiquitinated proteins by tandem mass spectrometry
Author(s) -
Vasilescu Julian,
Smith Jeffrey C.,
Zweitzig Daniel R.,
Denis Nicholas J.,
Haines Dale S.,
Figeys Daniel
Publication year - 2008
Publication title -
journal of mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.475
H-Index - 121
eISSN - 1096-9888
pISSN - 1076-5174
DOI - 10.1002/jms.1297
Subject(s) - chemistry , cutoff , mass spectrometry , tandem mass spectrometry , ubiquitin , false positive rate , chromatography , tandem , ion trap , analytical chemistry (journal) , ion , selected reaction monitoring , statistics , biochemistry , physics , mathematics , materials science , organic chemistry , quantum mechanics , composite material , gene
We report a simple approach for determining ion score cutoffs that permit the confident identification of ubiquitinated proteins by tandem mass spectrometry (MS/MS). Initial experiments involving the analysis of gel bands containing multi‐Ubiquitin chains with quadrupole time‐of‐flight and quadrupole ion trap mass spectrometers revealed that standard ion score cutoffs used for database searching were not sufficiently stringent. We also found that false positive and false negative rates (FPR and FNR) varied significantly depending on the cutoff scores used and that appropriate cutoffs could only be determined following a systematic evaluation of false positive rates. When standard cutoff scores were used for the analysis of complex mixtures of ubiquitinated proteins, unacceptably high FPR were observed. Finally, we found that FPR for ubiquitinated proteins are affected by the size of the protein database that is searched. These observations may be applicable for the study of other post‐translational modifications. Copyright © 2007 John Wiley & Sons, Ltd.