PhosphoHunter: An Efficient Software Tool for Phosphopeptide Identification

Phosphorylation is a protein posttranslational modification. It is responsible of the activation/inactivation of disease-related pathways, thanks to its role of “molecular switch.” The study of phosphorylated proteins becomes a key point for the proteomic analyses focused on the identification of diagnostic/therapeutic targets. Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) is the most widely used analytical approach. Although unmodified peptides are automatically identified by consolidated algorithms, phosphopeptides still require automated tools to avoid time-consuming manual interpretation. To improve phosphopeptide identification efficiency, a novel procedure was developed and implemented in a Perl/C tool called PhosphoHunter, here proposed and evaluated. It includes a preliminary heuristic step for filtering out the MS/MS spectra produced by nonphosphorylated peptides before sequence identification. A method to assess the statistical significance of identified phosphopeptides was also formulated. PhosphoHunter performance was tested on a dataset of 1500 MS/MS spectra and it was compared with two other tools: Mascot and Inspect. Comparisons demonstrated that a strong point of PhosphoHunter is sensitivity, suggesting that it is able to identify real phosphopeptides with superior performance. Performance indexes depend on a single parameter (intensity threshold) that users can tune according to the study aim. All the three tools localized >90% of phosphosites.