等电聚焦分离与^18O稳定同位素标记联用的磷酸化蛋白质组学定量方法研究

Quantitative Phosphoproteomics Methods Based on Isoelectric Focusing and ^(18)O Labeling Method

磷酸化蛋白质组学定量分析,要对磷酸化修饰富集技术和定量技术进行研究。基于此,本研究采用18O稳定同位素标记技术对胰蛋白酶酶解肽段混合物进行标记,并对其标记时间和标记后胰蛋白酶的变性条件进行优化。结果表明:在pH=4～5的KH2PO4缓冲体系中,37℃,标记反应持续19～24h,除了C-端肽之外,几乎所有的肽段都可达到100%标记;采用TCEP可以有效地抑制16O-18O回标现象。建立了与18O标记技术兼容性良好的IPG-IEF技术对磷酸化肽段进行选择性富集,富集后共从HepG2细胞中鉴定到491个磷酸化位点、362个磷酸化肽段和356个磷酸化蛋白,表明IPG-IEF在大规模磷酸化肽段分离富集中是有效的;最后与高准确度高灵敏度高分辨率的LTQ-FTICR质谱仪联用,建立了基于18O-IPG-IEF-LTQ-FTICR的磷酸化蛋白质组定量技术。实验结果表明,该技术可以实现磷酸化肽段的有效定性和定量。本研究为磷酸化蛋白质组学定量研究提供了实用技术。

For the analysis of quantitative phosphoproteomics, both qualitative and quantitative strategies are needed to be studied. Therefore, ^18O labeling method was used to label the tryptic phosphopeptides. The labeling conditions including the labeling time and the inactivation of the trypsin after labeling were optimized. The experimental results showed that the incorporation oxygen-18 isotopes for almost all peptides but the C-terminal peptides not ending on a lysine or an arginine could drive to 100% with tryptic catalyzing in the KH2PO4 buffer system （at pH = 4 -5 ） for 19 -24 h at 37℃. Tris （2-carboxyethyl）phosphine（TCEP） chosen as the inactivation of trypsin could effectively inhibit the exchange of incorporated oxygen-18 isotopes with oxygen-16 isotopes. Then the phosphopeptides enrichment technology isoelectric focusing on an immobilized pH gradient gel （IPG-IEF） was built, and 491 phosphosites, 362 phosphopeptides and 356 phosphoproteins were identified from HepG2 cells. This suggested that IPG-IEF was effective in the phosphopeptides enrichment analysis on a large scale and could be well compatible with ^18O labeling method. Finally, combining with linear ion trap Fourier transform ion cyclotron resonance （LTQ-FTICR） mass spectrometry, ^18O-IPG-IEF-LTQ-FTICR was built and demonstrated to be effective in qualitative and quantitative phosphoproteomics study by the experiment results.