Phosphoproteomics is a powerful analytical platform for identification and quantification of phosphorylated peptides and assignment of phosphorylation sites. Bioinformatics tools to identify phosphorylated peptides from their tandem mass spectra and protein sequence databases are important part of phosphoproteomics. In this work, we discuss general informatics aspects of mass-spectrometry-based phosphoproteomics. Some of the specifics of phosphopeptide identifications stem from the labile nature of phosphor groups and expanded peptide search space. Allowing for modifications of Ser, Thr, and Tyr residues exponentially increases effective database size. High mass resolution and accuracy measurements of precursor mass-to-charge ratios help to restrict the search space of candidate peptide sequences. The higher-order fragmentations of neutral loss ions enhance the fragment ion mass spectra of phosphorylated peptides. We show an example of a phosphopeptide identification where accounting for fragmentation from neutral loss species improves the identification scores in a database search algorithm by 50%. 1. Introduction The reversible phosphorylation of proteins regulates many aspects of cell life [1–3]. Phosphorylation and dephosphorylation, catalyzed by protein kinases and protein phosphatases, can change the function of a protein, for example, increase or decrease its biological activity, stabilize it or mark it for destruction, facilitate or inhibit movement between subcellular compartments, initiate or disrupt protein-protein interactions [1]. It is estimated that 30% of all cellular proteins are phosphorylated on at least one residue [4]. Abnormal phosphorylation is now recognized as a cause or consequence of many human diseases. Several natural toxins and tumor promoters produce their effects by targeting particular protein kinases [5, 6] and phosphatases. Protein kinases catalyze the transfer of the γ-phosphate from ATP to specific amino acids in proteins; in eukaryotes, these are usually Ser, Thr, and Tyr residues. Mass-spectrometry-based proteomics has emerged as a powerful platform for the analysis of protein phosphorylations [7]. In particular, the shotgun proteomics [8], using liquid chromatography coupled with mass spectrometry (LC-MS), has been successfully employed for comprehensive analysis of global phosphoproteome [6, 9, 10]. The advances in the phosphoproteomics were driven by developments in mass spectrometry (high resolution and mass accuracy), peptide/protein separation, phosphopeptide/protein enrichment, peptide fragmentation [11,
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