CAS Key Laboratory of Separation Sciences for Analytical Chemistry, National Chromatographic R&A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China.
Anal Chem. 2011 Oct 15;83(20):7755-62. doi: 10.1021/ac201299j. Epub 2011 Sep 21.
Accurately quantifying the changes of phosphorylation level on specific sites is crucial to understand the role of protein phosphorylation in physiological and pathological processes. Here, a pseudo triplex stable isotope dimethyl labeling approach was developed to improve the accuracy and the throughput of comprehensive quantitative phosphoproteome analyses. In this strategy, two identical samples are labeled with light and heavy isotopes, respectively, while another comparative sample is labeled with an intermediate isotope. Two replicated quantification results were achieved in just one experiment, and the relative standard deviation (RSD) criterion was used to control the quantification accuracy. Compared with the conventional duplex labeling approach, the number of quantified phosphopeptides increased nearly 50% and the experimental time was reduced by 50% under the same quantification accuracy. Combined with the automated online reversed phase-strong cation exchange-reversed phase (RP-SCX-RP) multidimensional separation system, a comparative phosphoproteome analysis of hepatocellular carcinoma (HCC) and normal human liver tissues was performed. Over 1800 phosphopeptides corresponding to ~2000 phosphorylation sites were quantified reliably in a 42 h multidimensional analysis. The pro-directed motifs, which were mainly associated with the extracellular signal-regulated kinases (ERKs), were observed as being overrepresented in the regulated phosphorylation sites, and some quantification results of phosphorylation sites were validated by the other studies. Therefore, this pseudo triplex labeling approach was demonstrated as a promising alternative for the comprehensive quantitative phosphoproteome analysis.
准确地定量测定特定位点的磷酸化水平对于理解蛋白质磷酸化在生理和病理过程中的作用至关重要。在这里，开发了一种拟三联稳定同位素双甲基标记方法，以提高综合定量磷酸化蛋白质组分析的准确性和通量。在该策略中，两个相同的样品分别用轻和重同位素标记，而另一个比较样品用中间同位素标记。仅在一个实验中就实现了两个重复的定量结果，并且使用相对标准偏差（RSD）标准来控制定量准确性。与传统的双标记方法相比，在相同的定量准确性下，定量的磷酸肽数量增加了近 50%，实验时间减少了 50%。结合自动化在线反相-强阳离子交换-反相（RP-SCX-RP）多维分离系统，对肝癌（HCC）和正常人类肝脏组织进行了比较磷酸化蛋白质组分析。在 42 小时的多维分析中，可靠地定量了超过 1800 种对应于~2000 个磷酸化位点的磷酸肽。正向导向基序主要与细胞外信号调节激酶（ERKs）相关，在调节磷酸化位点中被观察到过度表达，并且一些磷酸化位点的定量结果通过其他研究得到了验证。因此，这种拟三联体标记方法被证明是综合定量磷酸化蛋白质组分析的一种很有前途的替代方法。
