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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Stable isotope-free relative and absolute quantitation of protein phosphorylation stoichiometry by MS
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Stable isotope-free relative and absolute quantitation of protein phosphorylation stoichiometry by MS

机译：稳定的无同位素相对和绝对定量质谱分析蛋白质磷酸化化学计量

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Qualitative and quantitative information are crucial to a detailed understanding of the function of protein phosphorylation. MS is now becoming a quantitative approach to analyze protein phosphorylation. All methods that have been described either require the elaborate/expensive use of stable isotopes to compare a limited number of samples or do not provide phosphorylation stoichiometries. Here, we present stable isotope-free MS strategies that allow relative and absolute quantitation of phosphorylation stoichiometries. By using the developed methods, we can normalize to robustly account for run-to-run variations and variations in amounts of starting material. This procedure monitors the unmodified proteolytic peptides derived from the protein of interest and identifies peptides that are suitable for normalization purposes. Also, we can determine changes in phosphorylation stoichiometry by monitoring the changes in the normalized ion currents of the phosphopeptide(s) of interest. Absolute phosphorylation stoichiometry are measured by monitoring the ion currents of a phos-phopeptide and its unmodified cognate as the signal intensity changes of both peptide species are correlated. The method is applicable to multiply phosphorylated species (for which one more sample with varying phosphorylation stoichiometry than number of phosphorylation sites is required to correct for the differences in the ionization/detection efficiencies of the phosphopeptide, its partially phosphorylated and unphosphorylated cognates). Last, we can quantitate species with ragged ends resulting from incomplete proteolysis and measure phosphorylation stoichiometries of single samples by controlled dephosphorylation. These approaches were validated and subsequently applied to the phosphorylation of the yeast transcription factor Pho4.
机译：定性和定量信息对于详细了解蛋白质磷酸化功能至关重要。 MS现在正成为分析蛋白质磷酸化的定量方法。已描述的所有方法要么需要精心/昂贵地使用稳定同位素来比较有限数量的样品，要么不提供磷酸化化学计量。在这里，我们提出了稳定的无同位素质谱策略，可以相对和绝对定量磷酸化化学计量。通过使用开发的方法，我们可以进行归一化以可靠地说明每次运行之间的变化以及起始原料量的变化。该程序监测衍生自目的蛋白的未修饰蛋白水解肽，并鉴定适合标准化目的的肽。同样，我们可以通过监测目标磷酸肽的归一化离子电流的变化来确定磷酸化化学计量的变化。当两种肽的信号强度变化相关时，通过监测磷酸肽及其未修饰同源物的离子电流来测量绝对磷酸化化学计量。该方法适用于多种磷酸化物质（需要更多的具有比磷酸化位点数量更多的磷酸化化学计量的样品来校正磷酸肽，其部分磷酸化和未磷酸化的同源物的电离/检测效率的差异）。最后，我们可以对由于不完全蛋白水解导致末端参差不齐的物种进行定量，并通过控制去磷酸化来测量单个样品的磷酸化化学计量。这些方法已得到验证，随后应用于酵母转录因子Pho4的磷酸化。

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