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Computational phosphoproteomics: from identification to localization.

Lee DC, Jones AR, Hubbard SJ - Proteomics (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Life Sciences, University of Manchester, Manchester, UK.

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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).
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fig01: 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).

Mentions: Site localization appears to be a far more challenging task compared to identification, as demonstrated by the iPRG ABRF study, because unambiguous site localization relies on the presence of intact product ions in the product ion spectrum that are characteristic of a given candidate site. To make the problem even worse, site localization becomes considerably more difficult when candidate sites are found in close proximity in the peptide sequence, generating fewer discriminatory ions. As a final testament to difficulty of the problem, the ABRF study highlighted a case where even experienced manual curators could not agree with each other when given the same MS/MS spectrum and the known sequence, shown in Fig.1.


Computational phosphoproteomics: from identification to localization.

Lee DC, Jones AR, Hubbard SJ - Proteomics (2015)

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).
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4384807&req=5

fig01: 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).
Mentions: Site localization appears to be a far more challenging task compared to identification, as demonstrated by the iPRG ABRF study, because unambiguous site localization relies on the presence of intact product ions in the product ion spectrum that are characteristic of a given candidate site. To make the problem even worse, site localization becomes considerably more difficult when candidate sites are found in close proximity in the peptide sequence, generating fewer discriminatory ions. As a final testament to difficulty of the problem, the ABRF study highlighted a case where even experienced manual curators could not agree with each other when given the same MS/MS spectrum and the known sequence, shown in Fig.1.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Life Sciences, University of Manchester, Manchester, UK.

Show MeSH