SimPhospho: a software tool enabling confident phosphosite assignment

Abstract

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.

Fig. 1.

(A) Screenshot of SimPhospho displaying the main features of software: simulation options, including ion intensity values, peak match precision, types of ions used for simulation, data filtering switch, as well as output statistics and progress bar. (B) Optimization of intensity values of simulated peaks. To determine the optimal default parameters for SimPhospho, we tested different ion intensity combinations for phosphoric acid neutral loss ions and for intact ions compared to original fragment ion intensities in spectra of nonphosphorylated peptides. We saw the largest number of correctly assigned phosphosites at <1% FLR achieved with a combination of 50 and 50% intensities for intact ions and neutral loss ions for pS and pT, and 50% intensities for intact ions for pY. Data for 100 and 10% (pS, pT) and 100% (pY) are given for reference, as this combination was chosen for the prototype program (Suni et al., 2015). See Supplementary Figure S1 for other intensity combinations