Stable isotope-free relative and absolute quantitation of protein phosphorylation stoichiometry by MS
Stable isotope-free relative and absolute quantitation of protein phosphorylation stoichiometry by MS
复制标题
DOI：
10.1073/pnas.0409536102
复制
发表时间：
2005-03-15
影响因子：
11.1
通讯作者：
Kirschner, MW
中科院分区：
文献类型：
--
作者：
Steen, H;Jebanathirajah, JA;Kirschner, MW
定性和定量的信息对于详细了解蛋白质磷酸化的功能至关重要。MS现在正成为一种定量分析蛋白质磷酸化的方法。已经描述的所有方法要么需要复杂/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MS策略，允许相对和绝对定量的磷酸化化学计量。通过使用所开发的方法，我们可以归一化以稳健地解释运行间变化和起始材料量的变化。该程序监测来源于目的蛋白质的未纯化的蛋白水解肽，并鉴定适于标准化目的的肽。此外，我们可以通过监测感兴趣的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。通过监测磷酸肽及其未修饰的同源物的离子电流来测量绝对磷酸化化学计量，因为两种肽物质的信号强度变化是相关的。该方法适用于多重磷酸化物质（需要比磷酸化位点数量多一个具有不同磷酸化化学计量的样品，以校正磷酸肽、其部分磷酸化和未磷酸化同源物的电离/检测效率的差异）。最后，我们可以定量的物种与不完全的蛋白质水解和测量磷酸化化学计量的单一样品的控制去磷酸化。这些方法得到验证，并随后应用于磷酸化的酵母转录因子Pho 4。
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 unmodif ied 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 phosphopeptide 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.