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

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• 作者：Yasushi Ishihama ; Fan-Yan Wei ; Ken Aoshima ; Toshitaka Sato ; Junro Kuromitsu ; Yoshiya Oda
• 刊名：Journal of Proteome Research
• 出版年：2007
• 出版时间：March 2007
• 年：2007
• 卷：6
• 期：3
• 页码：1139 - 1144
• 全文大小：188K
• 年卷期：v.6,no.3(March 2007)
• ISSN：1535-3907

文摘

Immobilized metal affinity chromatography (IMAC) and titanium oxide (TiO₂) chromatography aresimple, widely used, and cost-effective methods to enrich phosphopeptides, but the sample loadingbuffer composition, desalting procedure, and control of loading amount are critical to avoid nonspecificinteractions and to achieve efficient phosphopeptide enrichment. Although the combination of MS3analysis and high-resolution mass spectrometry (MS) is helpful to identify phosphopeptides, the qualityof many MS/MS spectra having a neutral loss peak of phosphate is still too poor to allow sequenceidentification, and this results in many false-negative as well as false-positive identifications. Here, wepresent a novel strategy, which is based on the use of alkaline phosphatase to remove phosphatesand analysis of phospho/dephosphopeptide retention times to increase the reliability of identification.The use of phospho/dephosphopeptide retention time ratios allows the identification of phosphopeptideswith high confidence with the aid of a focused database of dephosphopeptides. This approach wasvery effective to identify multiple phophorylations in tryptic peptides. A 'true' phosphorylation dataset should contain about 90% phospho-Ser and a few percent phospho-Tyr, and this ratio can be usedas a quality criterion for evaluation of data sets. By applying this efficient approach, we were able toidentify more than one thousand phosphopeptides.