A hierarchical MS²/MS³ database search algorithm for automated analysis of phosphopeptide tandem mass spectra

A novel hierarchical MS²/MS³ database search algorithm has been developed to analyze MS²/ MS³ phosphopeptides proteomic data. The algorithm is incorporated in an automated database search program, MassMatrix. The algorithm matches experimental MS ² spectra against a supplied protein database to determine candidate peptide matches. It then matches the corresponding experimental MS³ spectra against those candidate peptide matches. The MS² and MS ³ spectra are used in concert to arrive at peptide matches with overall higher confidence rather than combining MS² and MS ³ data searched separately. Receiver operating characteristic analysis showed that hierarchical MS²/MS³ database searches with MassMatrix had better sensitivity and specificity than the two-stage MS²/MS³ database searches obtained with MassMatrix, MASCOT, and X!Tandem. A greater number of true peptide matches at a given false rate were identified by use of this new algorithm for data collected on both LCQ and LTQ-FTICR mass spectrometers. The additional MS³ spectral data also improved the overall reliability and the number of true positives (TPs) due to the fact that the TPs of the MS²/MS ³ search results had higher scores than those of the MS².