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Advances in Bioinformatics
Volume 2015 (2015), Article ID 382869, 12 pages
Research Article

PhosphoHunter: An Efficient Software Tool for Phosphopeptide Identification

Dipartimento di Ingegneria Industriale e dell’Informazione, Università degli Studi di Pavia, Via Ferrata 5, 27100 Pavia, Italy

Received 15 October 2014; Revised 14 December 2014; Accepted 15 December 2014

Academic Editor: Ming Chen

Copyright © 2015 Alessandra Tiengo et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


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.