Computational phosphoproteomics: from identification to localization

Abstract

Analysis of the phosphoproteome by MS has become a key technology for the characterization of dynamic regulatory processes in the cell, since kinase and phosphatase action underlie many major biological functions. However, the addition of a phosphate group to a suitable side chain often confounds informatic analysis by generating product ion spectra that are more difficult to interpret (and consequently identify) relative to unmodified peptides. Collectively, these challenges have motivated bioinformaticians to create novel software tools and pipelines to assist in the identification of phosphopeptides in proteomic mixtures, and help pinpoint or "localize" the most likely site of modification in cases where there is ambiguity. Here we review the challenges to be met and the informatics solutions available to address them for phosphoproteomic analysis, as well as highlighting the difficulties associated with using them and the implications for data standards.

Keywords: Bioinformatics; Data processing and analysis; Phosphoproteomics; Technology.

Figure 1

Ambiguity in site assignment of phosphopeptides. The phosphopeptide above generates a product ion spectrum from which it is challenging to unambiguously determine the true site determining ions. In this particular case, two b ions highlighted in green boxes are consistent with serine at position 7 in the peptide being modified, or alternately, the threonine at position 9 could be modified yielding a characteristic y9 ion (green box, lower panel). Experts inspecting the spectrum were divided on which is the most likely interpretation. The possibility that both peptides were present is also not excluded, since they would have the same precursor ion m/z value (figure adapted from ABRF web site, http://www.abrf.org/index.cfm/group.show/ProteomicsInformaticsResearchGroup.53.htm).