1P-06 PDF
Optimization of SimSpectraST search conditions for phosphoproteomics
To perform qualitative and quantitative phosphoproteomics by mass spectrometry, it is very important to identify which amino acid residue is phosphorylated. A recently developed data analysis method SimSpectraST, which simulates beam-type CID spectra of phosphopeptides and perform spectral library searching using SpectraST, facilitates accurate and sensitive localization of phosphorylation sites. In this study, to improve the localization sensitivity, we have optimized SimSpectraST search conditions.
Orbitrap HCD data of synthetic monophosphorylated peptides and Hela phosphorylated peptides were used as test datasets. An HCD spectral library of simulated monophosphorylated peptides was used for spectral library searching with SpectraST version 5. Using the data of synthetic peptides with known phosphorylation sites, the discriminant score of SpectraST (F-value) giving 1% false localization rate (FLR) was determined. Hela phosphopeptides were identified at 1% false discovery rate (FDR), and the F-value for 1% FLR was applied. To optimize the SimSpectraST searching, four conditions were changed from the default and the obtained results were compared. Consequently, reduced mass tolerances for both precursor and fragment ions resulted in increased identifications of the Hela phosphopeptides at 1% FDR and 1% FLR. Currently we are investigating the compatibility of SimSpectraST searching with TripleTOF data.