Wang Hui, Duan Jicheng, Zhang Lihua, Liang Zhen, Zhang Weibing, Zhang Yukui
National Chromatographic Research & Analysis Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China.
J Sep Sci. 2008 Feb;31(3):480-7. doi: 10.1002/jssc.200700445.
The detection of phosphopeptides, especially multi-phosphopeptides, by tandem electrospray ionization mass spectrometry (ESI-MS/MS) is a great challenge due to their low abundance and the poor ionization efficiency of samples. In our recent study, a strategy was proposed for the analysis of trace multi-phosphopeptides which combined selective enrichment of phosphorylated peptides by TiO2 and dephosphorylation by alkaline phosphatase (AP). After separation by muHPLC, the profiles of enriched peptides before and after AP treatment were compared, and the additional peaks appearing in the latter case hinted at the existence of multi-phosphopeptides. Subsequently, an incomplete dephosphorylation reaction was performed to partially remove the phosphate groups so that the phosphorylation sites of the multi-phosphopeptides might be estimated. Through analysis of the digests of beta-casein and extracted proteins of bovine milk, more information on the multi-phosphopeptides was obtained by muHPLC-ESI-MS/MS than that obtained without AP treatment, which demonstrated that such a strategy might supply some potential information about trace multi-phosphopeptides lost in shotgun analysis.
通过串联电喷雾电离质谱（ESI-MS/MS）检测磷酸化肽段，尤其是多磷酸化肽段，是一项巨大的挑战，因为它们丰度低且样品电离效率差。在我们最近的研究中，提出了一种分析痕量多磷酸化肽段的策略，该策略将通过二氧化钛（TiO2）对磷酸化肽段进行选择性富集与碱性磷酸酶（AP）去磷酸化相结合。通过μHPLC分离后，比较了AP处理前后富集肽段的图谱，后一种情况下出现的额外峰暗示了多磷酸化肽段的存在。随后，进行不完全去磷酸化反应以部分去除磷酸基团，从而可以估计多磷酸化肽段的磷酸化位点。通过对β-酪蛋白的酶解产物和牛乳提取蛋白的分析，与未进行AP处理相比，μHPLC-ESI-MS/MS获得了更多关于多磷酸化肽段的信息，这表明该策略可能提供一些关于鸟枪法分析中丢失的痕量多磷酸化肽段的潜在信息。
