Optimization of TripleTOF spectral simulation and library searching for confident localization of phosphorylation sites.

Tandem mass spectrometry (MS/MS) has been used in analysis of proteins and their post-translational modifications. A recently developed data analysis method, which simulates MS/MS spectra of phosphopeptides and performs spectral library searching using SpectraST, facilitates confident localization of phosphorylation sites. However, its performance has been evaluated only on MS/MS spectra acquired using Orbitrap HCD mass spectrometers so far. In this study, we have investigated whether this approach would be applicable to another type of mass spectrometers, and optimized the simulation and search conditions to achieve sensitive and confident site localization. Synthetic phosphopeptides and enriched K562 cell phosphopeptides were analyzed using a TripleTOF 6600 mass spectrometer before and after enzymatic dephosphorylation. Dephosphorylated peptides identified by X!Tandem database searching were subjected to spectral simulation of all possible single phosphorylations using SimPhospho software. Phosphopeptides were identified and localized by SpectraST searching against a library of the simulated spectra. Although no synthetic phosphopeptide was localized at 1% false localization rate under the previous conditions, optimization of the spectral simulation and search conditions for the TripleTOF datasets achieved the localization and improved the sensitivity. Furthermore, the optimized conditions enabled sensitive localization of K562 phosphopeptides at 1% false discovery and localization rates. These results suggest that accurate phosphopeptide simulation of TripleTOF MS/MS spectra is possible and the simulated spectral libraries can be used in SpectraST searching for confident localization of phosphorylation sites.

SimPhospho: a software tool enabling confident phosphosite assignment.

Motivation: Mass spectrometry combined with enrichment strategies for phosphorylated peptides has been successfully employed for two decades to identify sites of phosphorylation. However, unambiguous phosphosite assignment is considered challenging. Given that site-specific phosphorylation events function as different molecular switches, validation of phosphorylation sites is of utmost importance. In our earlier study we developed a method based on simulated phosphopeptide spectral libraries, which enables highly sensitive and accurate phosphosite assignments. To promote more widespread use of this method, we here introduce a software implementation with improved usability and performance. Results: We present SimPhospho, a fast and user-friendly tool for accurate simulation of phosphopeptide tandem mass spectra. Simulated phosphopeptide spectral libraries are used to validate and supplement database search results, with a goal to improve reliable phosphoproteome identification and reporting. The presented program can be easily used together with the Trans-Proteomic Pipeline and integrated in a phosphoproteomics data analysis workflow. Availability and implementation: SimPhospho is open source and it is available for Windows, Linux and Mac operating systems. The software and its user's manual with detailed description of data analysis as well as test data can be found at https://sourceforge.net/projects/simphospho/. Supplementary information: Supplementary data are available at Bioinformatics online.