基于TMT10-plex等量标记结合SMOAC富集磷酸肽的定量磷酸化蛋白质组性能评价

探索并建立了一种快速、 简便且高通量定量磷酸化蛋白质组的策略, 即采用连续互补的磷酸化富集方法SMOAC(Sequential enrichment of metal oxide affinity chromatography)结合TMT(Tandem mass tag)标记技术定量磷酸化蛋白质组学. 以3例经紫草素处理的及3例正常的人肝癌 HepG2 细胞为实验材料, 经Trypsin酶解后的肽段用TMT10-plex试剂进行等量标记, 标记肽段先经TiO₂富集, 收集包含磷酸化肽段的洗脱液, 接着用次氮基三醋酸铁(Fe-NTA)对TiO₂的流穿液和清洗液进行二次富集, 再次收集包含磷酸化肽段的洗脱液. 整个实验流程做2组, 对其中一组的2次洗脱液分别分析, 另一组的2次洗脱液合并分析. 在SMOAC的2次洗脱液合并分析中鉴定到4263个磷酸化蛋白上超过13000条磷酸化肽, 富集特异性>97%, 其中被定量的磷酸化蛋白为3848个, 占总鉴定量的90%以上. 研究结果表明, SMOAC 能够有效提高磷酸化肽段的鉴定效率, 且能与TMT等量标记试剂结合, 实现对少量蛋白样品的磷酸化蛋白定量分析.

Evaluation of Quantitative Phosphoproteomic Performance of Sequential Enrichment of Metal Oxide Affinity Chromatography for Phosphopeptides Using TMT 10-plex Isobaric Labeling

A rapid， simple and high-flux strategy for quantitative phosphorylated proteomics was explored and developed， which was a kind of sequential enrichment method for complementary phosphopeptides labeled with Tandem mass tag（TMT） reagents. We cultured HepG2 cells treated with alkannin for 24 h and normal HepG2 cells for phosphorylation modification analysis. All the proteins were digested fully with Trypsin and labeled with TMT reagents， followed by desalting and vacuum drying. We implemented a SMOAC（Sequential enrichment of metal oxide affinity chromatography） strategy. We used firstly titanium dioxide（TiO₂） chromatography for phosphopeptides enrichment. Flow-through and wash fractions from TiO₂ were pooled and enriched secondly by ferrie nitrilotriacetate（Fe-NTA） chromatography. The platform equipped with EASY-nLC1200 and Orbitrap Exploris 480 mass spectrometry was applied to acquiring the phosphopeptides data of the HepG2 cells. We implemented two parallel sets of SMOAC strategy for 600 μg hybrid peptides labeled with TMT reagents. One of the sets， we pooled two kinds of eluate from SMOAC strategy for mass spectrometry analysis. Another of the sets， we analyzed the two kinds of eluate with mass spectrometry， respectively. Altogether we identified 4263 proteins and 16335 phosphopeptides， including 13347 phosphopeptides and 13851 phosphosites of 3848 proteins common to all channels from pooled eluate of SMOAC strategy. The specificity of the enrichment strategy was larger than 97% and 90% of the phosphoproteins identified were quantified. The study reveals SMOAC effectively improves the identification efficiency of phosphorylated peptides and also can quantify phosphorylation protein of a small amount of samples， combining with TMT reagents.