Efficient Identification of Phosphorylation by Mass Spectrometric Phosphopeptide Fingerprinting

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• 作者：Eileen M. Woo ; David Fenyo ; Benjamin H. Kwok ; Hironori Funabiki ; Brian T. Chait
• 刊名：Analytical Chemistry
• 出版年：2008
• 出版时间：April 1, 2008
• 年：2008
• 卷：80
• 期：7
• 页码：2419 - 2425
• 全文大小：211K
• 年卷期：v.80,no.7(April 1, 2008)
• ISSN：1520-6882

文摘

We describe a rapid and efficient method for the identification of phosphopeptides, which we term mass spectrometric (MS) phosphopeptide fingerprinting. The methodinvolves quantitative comparison of proteolytic peptidesfrom native versus completely dephosphorylated proteins.Dephosphorylation of serine, threonine, and tyrosineresidues is achieved by in-gel treatment of the separatedproteins with hydrogen fluoride (HF). This chemicaldephosphorylation results in enrichment of those unmodified peptides that correspond to previously phosphorylated peptides. Quantitative comparison of the signal-to-noise ratios of peaks in the treated versus untreatedsamples are used to identify phosphopeptides, which canbe confirmed and further studied by tandem mass spectrometry (MS/MS). We have applied this method toidentify eight known phosphorylation sites of XenopusAurora A kinase, as well as several novel sites in theXenopus chromosome passenger complex (CPC).