title: Systematic determination of ion score cutoffs based on calculated false positive rates: application for identifying ubiquitinated proteins by tandem mass spectrometry.
abstract: 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.
authors.affiliation: Ottawa Institute of Systems Biology, University of Ottawa, 451 Smyth Road, Ottawa, ON, K1H 8M5, Canada.
authors.forename: Julian
authors.initials: J
authors.lastname: Vasilescu
authors.forename: Jeffrey C
authors.initials: JC
authors.lastname: Smith
authors.forename: Daniel R
authors.initials: DR
authors.lastname: Zweitzig
authors.forename: Nicholas J
authors.initials: NJ
authors.lastname: Denis
authors.forename: Dale S
authors.initials: DS
authors.lastname: Haines
authors.forename: Daniel
authors.initials: D
authors.lastname: Figeys
mesh_terms: D045744:Cell Line, Tumor; D019295:Computational Biology; D030562:Databases, Protein; D005188:False Negative Reactions; D005189:False Positive Reactions; D006801:Humans; D007477:Ions / Q000032:analysis / Q000737:chemistry; D010455:Peptides / Q000032:analysis / Q000737:chemistry; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D053719:Tandem Mass Spectrometry / Q000379:methods* / Q000706:statistics & numerical data; D014357:Trypsin / Q000737:chemistry; D025801:Ubiquitin / Q000032:analysis / Q000737:chemistry; D054875:Ubiquitination
chemical_list: D007477:Ions; D010455:Peptides; D011506:Proteins; D025801:Ubiquitin; D014357:Trypsin