基于强阳离子交换色谱与等电聚焦的磷酸化蛋白质组学分离策略比较

Comparison of Phosphoproteomic Separation Strategies Based on Strong Cation Exchange Chromatography-Isoelectric Focusing Techniques

比较分析了强阳离子交换(SCX)与等电聚焦(IPG-IEF)技术在磷酸化蛋白质组学中的应用。采用3种标准磷酸化蛋白对SCX与IPG-IEF两种技术对磷酸化肽段富集的有效性进行考察。以HepG2细胞为复杂样本,考察SCX与IPG-IEF在实际样本中的应用情况。对SCX与IPG-IEF技术在18O标记的磷酸化蛋白质组定量研究中的应用情况进行考察。蛋白鉴定采用高准确度、高灵敏度、高分辨率的LTQ-FTICR-MS/MS质谱仪。实验表明:SCX和IPG-IEF在大规模磷酸化肽段分离过程中均有效;在复杂样本中,SCX技术的分离效果优于IPG-IEF;IPG-IEF的重复性好于SCX;在磷酸化蛋白质组定量分析结果表明,IPG-IEF技术的稳定性优于SCX。本研究为根据不同实验目的而选择适当的磷酸化蛋白质组预分离技术提供了有用信息。

Efficient pre-purification steps for the enrichment of phosphorylated proteins or phosphopeptides are necessary for better detection of phosphorylation sites in phosphoproteomic analysis.Currently,the most common first-dimensional separation technique used is strong cation exchange（SCX）.A potential alternative to SCX-based separation is to use isoelectric focusing（IEF） as a first-dimensional separation technique,which has been demonstrated recently.In this study,we present a direct comparison between SCX and IEF based on IPG strips（IPG-IEF） for the phosphoproteomic separation.The comparison experiments discussed in this study utilized standard phosphoproteins and a real sample of HepG2 cell.Then the comparison of 18O labeling phosphopeptides′ stability under immobilized pH gradient gel（IPG）-IEF with under SCX was made.Fractions from both technique（SCX and IPG-IEF） were analyzed using the High Mass Accuracy LTQ-FTICR-MS/MS.The results demonstrate that SCX-LTQ-FT and IPG-IEF-LTQ-FT are useful in the phosphopeptides enrichment analysis on a large scale.And SCX-LTQ-FT is relative superior to IPG-IEF-LTQ-FT,whereas the 18O labeling phosphopeptides′stability under SCX-LTQ-FT is relatively poor with that under IPG-IEF-LTQ-FT.