Quantifying Kinase-Specific Phosphorylation Stoichiometry Using Stable Isotope Labeling In a Reverse In-Gel Kinase Assay

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• 作者：Xiang Li ; Jonathan T. Cox ; Weiliang Huang ; Maureen Kane ; Keqi Tang ; Charles J. Bieberich
• 刊名：Analytical Chemistry
• 出版年：2016
• 出版时间：December 6, 2016
• 年：2016
• 卷：88
• 期：23
• 页码：11468-11475
• 全文大小：367K
• ISSN：1520-6882

文摘

Despite recent advancements in large-scale phosphoproteomics, methods to quantify kinase-specific phosphorylation stoichiometry of protein substrates are lacking. We developed a method to quantify kinase-specific phosphorylation stoichiometry by combining the reverse in-gel kinase assay (RIKA) with high-resolution liquid chromatography–mass spectrometry (LC–MS). Beginning with predetermined ratios of phosphorylated to nonphosphorylated protein kinase CK2 (CK2) substrate molecules, we employed ¹⁸O-labeled adenosine triphosphate (¹⁸O-ATP) as the phosphate donor in a RIKA, then quantified the ratio of ¹⁸O- versus ¹⁶O-labeled tryptic phosphopeptide using high mass accuracy mass spectrometry (MS). We demonstrate that the phosphorylation stoichiometry determined by this method across a broad percent phosphorylation range correlated extremely well with the predicted value (correlation coefficient = 0.99). This approach provides a quantitative alternative to antibody-based methods of determining the extent of phosphorylation of a substrate pool.