Protein and Proteome Phosphorylation Stoichiometry Analysis by Element Mass Spectrometry

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• 作者：Ralf Krü ; ger ; Dieter Kü ; bler ; Roser Pallissé ; Andreas Burkovski ; and Wolf D. Lehmann
• 刊名：Analytical Chemistry
• 出版年：2006
• 出版时间：March 15, 2006
• 年：2006
• 卷：78
• 期：6
• 页码：1987 - 1994
• 全文大小：353K
• 年卷期：v.78,no.6(March 15, 2006)
• ISSN：1520-6882

文摘

Protein phosphorylation stoichiometry was assessed bytwo analytical strategies. Both are based on element massspectrometry (ICPMS, inductively coupled plasma massspectrometry) and simultaneous monitoring of ³¹P and³⁴S. One strategy employs a combination of 1D gelelectrophoresis, in-gel digestion, and final LC-ICPMSanalysis (LC = capillary liquid chromatography). Theother strategy uses the combination of 1D gel electrophoresis, protein blotting, and imLA-ICPMS (imLA =imaging laser ablation). The two methods were evaluatedwith standard phosphoproteins and were applied to theanalysis of the cytoplasmatic proteome of bacterial cells(Corynebacterium glutamicum) and eukaryotic cells(Mus musculus). The eukaryotic proteome was found toexhibit a significantly higher phosphorylation degree(~0.8 mol of P/mol of protein) compared to the bacterialproteome (~0.01 mol of P/mol of protein). Both analyticalstrategies revealed consistent quantitative results, with theLC-ICPMS approach providing the higher sensitivity.In summary, two ICPMS-based methods for quantitativeestimation of the phosphorylation degree of a cellularproteome are presented which access the native proteomestate and do not require any type of label introduction orderivatization.