title: Universal and confident phosphorylation site localization using phosphoRS.
abstract: An algorithm for the assignment of phosphorylation sites in peptides is described. The program uses tandem mass spectrometry data in conjunction with the respective peptide sequences to calculate site probabilities for all potential phosphorylation sites. Tandem mass spectra from synthetic phosphopeptides were used for optimization of the scoring parameters employing all commonly used fragmentation techniques. Calculation of probabilities was adapted to the different fragmentation methods and to the maximum mass deviation of the analysis. The software includes a novel approach to peak extraction, required for matching experimental data to the theoretical values of all isoforms, by defining individual peak depths for the different regions of the tandem mass spectrum. Mixtures of synthetic phosphopeptides were used to validate the program by calculation of its false localization rate versus site probability cutoff characteristic. Notably, the empirical obtained precision was higher than indicated by the applied probability cutoff. In addition, the performance of the algorithm was compared to existing approaches to site localization such as Ascore. In order to assess the practical applicability of the algorithm to large data sets, phosphopeptides from a biological sample were analyzed, localizing more than 3000 nonredundant phosphorylation sites. Finally, the results obtained for the different fragmentation methods and localization tools were compared and discussed.
authors.affiliation: Research Institute of Molecular Pathology, Dr. Bohrgasse 7, A-1030 Vienna, Austria.
authors.forename: Thomas
authors.initials: T
authors.lastname: Taus
authors.forename: Thomas
authors.initials: T
authors.lastname: Köcher
authors.forename: Peter
authors.initials: P
authors.lastname: Pichler
authors.forename: Carmen
authors.initials: C
authors.lastname: Paschke
authors.forename: Andreas
authors.initials: A
authors.lastname: Schmidt
authors.forename: Christoph
authors.initials: C
authors.lastname: Henrich
authors.forename: Karl
authors.initials: K
authors.lastname: Mechtler
mesh_terms: D000465:Algorithms; D001665:Binding Sites; D005591:Chemical Fractionation / Q000379:methods; D006367:HeLa Cells; D006801:Humans; D010748:Phosphopeptides / Q000138:chemical synthesis / Q000737:chemistry* / Q000302:isolation & purification; D010766:Phosphorylation; D020033:Protein Isoforms / Q000737:chemistry; D040901:Proteomics / Q000379:methods* / Q000592:standards; D015203:Reproducibility of Results; D012984:Software; D053719:Tandem Mass Spectrometry
chemical_list: D010748:Phosphopeptides; D020033:Protein Isoforms