Dataset Information

Estimating the Efficiency of Phosphopeptide Identification by Tandem Mass Spectrometry.

ABSTRACT: Mass spectrometry has played a significant role in the identification of unknown phosphoproteins and sites of phosphorylation in biological samples. Analyses of protein phosphorylation, particularly large scale phosphoproteomic experiments, have recently been enhanced by efficient enrichment, fast and accurate instrumentation, and better software, but challenges remain because of the low stoichiometry of phosphorylation and poor phosphopeptide ionization efficiency and fragmentation due to neutral loss. Phosphoproteomics has become an important dimension in systems biology studies, and it is essential to have efficient analytical tools to cover a broad range of signaling events. To evaluate current mass spectrometric performance, we present here a novel method to estimate the efficiency of phosphopeptide identification by tandem mass spectrometry. Phosphopeptides were directly isolated from whole plant cell extracts, dephosphorylated, and then incubated with one of three purified kinases-casein kinase II, mitogen-activated protein kinase 6, and SNF-related protein kinase 2.6-along with 16O4- and 18O4-ATP separately for in vitro kinase reactions. Phosphopeptides were enriched and analyzed by LC-MS. The phosphopeptide identification rate was estimated by comparing phosphopeptides identified by tandem mass spectrometry with phosphopeptide pairs generated by stable isotope labeled kinase reactions. Overall, we found that current high speed and high accuracy mass spectrometers can only identify 20%-40% of total phosphopeptides primarily due to relatively poor fragmentation, additional modifications, and low abundance, highlighting the urgent need for continuous efforts to improve phosphopeptide identification efficiency. Graphical Abstract ?.

SUBMITTER: Hsu CC

PROVIDER: S-EPMC5438756 | biostudies-literature | 2017 Jun

REPOSITORIES: biostudies-literature

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Estimating the Efficiency of Phosphopeptide Identification by Tandem Mass Spectrometry.

Hsu Chuan-Chih CC Xue Liang L Arrington Justine V JV Wang Pengcheng P Paez Paez Juan Sebastian JS Zhou Yuan Y Zhu Jian-Kang JK Tao W Andy WA

Journal of the American Society for Mass Spectrometry 20170310 6

Mass spectrometry has played a significant role in the identification of unknown phosphoproteins and sites of phosphorylation in biological samples. Analyses of protein phosphorylation, particularly large scale phosphoproteomic experiments, have recently been enhanced by efficient enrichment, fast and accurate instrumentation, and better software, but challenges remain because of the low stoichiometry of phosphorylation and poor phosphopeptide ionization efficiency and fragmentation due to neutr ...[more]

PMID: 28283928

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Project description:MotivationComplex patterns of protein phosphorylation mediate many cellular processes. Tandem mass spectrometry (MS/MS) is a powerful tool for identifying these post-translational modifications. In high-throughput experiments, mass spectrometry database search engines, such as MASCOT provide a ranked list of peptide identifications based on hundreds of thousands of MS/MS spectra obtained in a mass spectrometry experiment. These search results are not in themselves sufficient for confident assignment of phosphorylation sites as identification of characteristic mass differences requires time-consuming manual assessment of the spectra by an experienced analyst. The time required for manual assessment has previously rendered high-throughput confident assignment of phosphorylation sites challenging.ResultsWe have developed a knowledge base of criteria, which replicate expert assessment, allowing more than half of cases to be automatically validated and site assignments verified with a high degree of confidence. This was assessed by comparing automated spectral interpretation with careful manual examination of the assignments for 501 peptides above the 1% false discovery rate (FDR) threshold corresponding to 259 putative phosphorylation sites in 74 proteins of the Trypanosoma brucei proteome. Despite this stringent approach, we are able to validate 80 of the 91 phosphorylation sites (88%) positively identified by manual examination of the spectra used for the MASCOT searches with a FDR < 15%.ConclusionsHigh-throughput computational analysis can provide a viable second stage validation of primary mass spectrometry database search results. Such validation gives rapid access to a systems level overview of protein phosphorylation in the experiment under investigation.AvailabilityA GPL licensed software implementation in Perl for analysis and spectrum annotation is available in the supplementary material and a web server can be assessed online at http://www.compbio.dundee.ac.uk/prophossi.

Dataset Information

Estimating the Efficiency of Phosphopeptide Identification by Tandem Mass Spectrometry.

Publications

Estimating the Efficiency of Phosphopeptide Identification by Tandem Mass Spectrometry.

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