Translational Medicine Research Collaboration, Dundee DD1 9SY, United Kingdom.
J Proteome Res. 2010 Feb 5;9(2):743-51. doi: 10.1021/pr900572h.
The Ptdlns-3-kinase (PI3-K) signaling pathway plays a vital role in cell survival, proliferation, apoptosis and differentiation in normal cells, as well as in diseases such as cancer and diabetes. Quantification of phospho-Akt is a standard way of assessing the activity of the PI3-K signaling pathway in both cells and tumors. This measurement is traditionally performed semiquantitatively using immunoassays such as Western blot. Here we report an LC-MS method to accurately measure the stoichiometry of Akt phosphorylation in biological samples. The procedure includes immunoprecipitation, gel electrophoresis, in-gel digestion, addition of isotopicaly labeled internal standards and LC-MS/MS. Two proteolytic enzymes, chymotrypsin and trypsin, were used to generate suitable peptide fragments for measuring Thr308 and Ser473 phosphorylation, respectively. The interday imprecision was estimated to be 3.8% and 2.3% for Thr308 and Ser473, respectively. This method has been tested on human T-cells grown in presence and absence of pervanadate and with or without a PI3-K inhibitor and on human glioblastoma cells (U-87 MG) grown in presence and absence of wortmannin (PI3-K inhibitor).The results of T cells suggest that the levels of Akt phosphorylation in untreated cells were below 1% for both phosphorylation sites. Pervanadate treatment provoked an 18-fold increase in phosphorylation of Thr308 and the PI3-K inhibitor partially reversed the increase. A comparison between LC-MS/MS and Western blotting suggests that the LC-MS based method is of comparable sensitivity and provides a more accurate phosphorylation stoichiometry, a wider dynamic range and more in-depth information. The application of the new method and its utility to providing predictive markers of response to targeted therapies is discussed.
磷脂酰肌醇-3-激酶（PI3-K）信号通路在正常细胞的细胞存活、增殖、凋亡和分化以及癌症和糖尿病等疾病中起着至关重要的作用。磷酸化 Akt 的定量分析是评估 PI3-K 信号通路在细胞和肿瘤中活性的一种标准方法。这种测量传统上是使用免疫测定法（如 Western blot）进行半定量分析。在这里，我们报告了一种 LC-MS 方法，可以准确测量生物样品中 Akt 磷酸化的化学计量。该程序包括免疫沉淀、凝胶电泳、胶内消化、添加同位素标记的内标和 LC-MS/MS。两种蛋白水解酶，胰凝乳蛋白酶和胰蛋白酶，分别用于生成适合测量 Thr308 和 Ser473 磷酸化的肽片段。Thr308 和 Ser473 的日内精密度分别估计为 3.8%和 2.3%。该方法已在存在和不存在过钒酸盐以及存在和不存在 PI3-K 抑制剂的情况下培养的人 T 细胞以及在存在和不存在wortmannin（PI3-K 抑制剂）的情况下培养的人神经胶质瘤细胞（U-87 MG）上进行了测试。T 细胞的结果表明，未经处理的细胞中 Akt 磷酸化水平在两个磷酸化位点均低于 1%。过钒酸盐处理引起 Thr308 磷酸化增加 18 倍，PI3-K 抑制剂部分逆转了这种增加。LC-MS/MS 与 Western blot 的比较表明，基于 LC-MS 的方法具有相当的灵敏度，并提供更准确的磷酸化化学计量、更宽的动态范围和更深入的信息。讨论了新方法的应用及其在提供针对靶向治疗的反应预测标志物方面的实用性。
