As with all shotgun proteomics experiments, global quantitative phosphoproteomics relies heavily on appropriate bioinformatics analyses. The relevant bioinformatics methods associated with global quantitative phosphoproteomics are confident identification and validation of thousands of phosphopeptides from MS/MS spectra, determination of phosphorylation stoichiometry of phosphopeptides, localization of phosphorylation sites, and measurement of the ratio of phosphorylated peptides. Each one of these steps is of equal importance. That is, if any one of these steps is inaccurate or of low confidence, the entire quantitative analysis is equally inaccurate and of low confidence.

Identification of phosphopeptide sequences and measurement of phosphorylated peptides leverages the accuracy of global quantitative proteomics (i.e. SEQUEST, ProLuCID, DTASelect, and CENSUS). When the appropriate filtering methods are used, these two steps are already of high confidence. The remaining essential steps of phosphosphopeptide validation, determination of phosphorylation stoichiometry of phosphopeptides and phosphorylation site localization have been addressed by the introduction of Debunker and AScore.

This section of the IP2 user manual describes the integration and usage of Debunker and Ascore in the quantitative pipeline (Phospho Quant) of IP2 using Orbitrap MS data and 15N isotopic labeling. These methods can be applied to other high mass accuracy instruments (i.e. TOF, FTIRC, etc.) and labeling strategies (e.g. SILAC).

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