Enhancement of the Efficiency of Phosphoproteomic Identification by Removing Phosphates after Phosphopeptide Enrichment

Citations Over TimeTop 10% of 2007 papers

Abstract

Immobilized metal affinity chromatography (IMAC) and titanium oxide (TiO2) chromatography are simple, widely used, and cost-effective methods to enrich phosphopeptides, but the sample loading buffer composition, desalting procedure, and control of loading amount are critical to avoid nonspecific interactions and to achieve efficient phosphopeptide enrichment. Although the combination of MS3 analysis and high-resolution mass spectrometry (MS) is helpful to identify phosphopeptides, the quality of many MS/MS spectra having a neutral loss peak of phosphate is still too poor to allow sequence identification, and this results in many false-negative as well as false-positive identifications. Here, we present a novel strategy, which is based on the use of alkaline phosphatase to remove phosphates and analysis of phospho/dephosphopeptide retention times to increase the reliability of identification. The use of phospho/dephosphopeptide retention time ratios allows the identification of phosphopeptides with high confidence with the aid of a focused database of dephosphopeptides. This approach was very effective to identify multiple phophorylations in tryptic peptides. A 'true' phosphorylation data set should contain about 90% phospho-Ser and a few percent phospho-Tyr, and this ratio can be used as a quality criterion for evaluation of data sets. By applying this efficient approach, we were able to identify more than one thousand phosphopeptides.

Related Papers

• → Highly Robust, Automated, and Sensitive Online TiO₂-Based Phosphoproteomics Applied To Study Endogenous Phosphorylation in Drosophila melanogaster(2007)168 cited
• → Rapid and Reproducible Single-Stage Phosphopeptide Enrichment of Complex Peptide Mixtures: Application to General and Phosphotyrosine-Specific Phosphoproteomics Experiments(2011)155 cited
• → Characterization of a TiO2 enrichment method for label-free quantitative phosphoproteomics(2011)114 cited
• → Reference-facilitated Phosphoproteomics(2007)74 cited
• → An integrated global strategy for cell lysis, fractionation, enrichment and mass spectrometric analysis of phosphorylated peptides(2010)38 cited