基于Ti^(4+)-IMAC富集的分枝菌酸小杆菌深度覆盖磷酸化蛋白质组研究

In-depth characterization of Mycolicibacterium smegmatis MC^(2)155 phosphoproteome based on the Ti^(4+)-IMAC enrichment strategy

【目的】本研究以分枝菌酸小杆菌(Mycolicibacterium smegmatis)为研究对象,探索适于原核微生物理想的磷酸化富集方法。【方法】我们比较了二氧化钛(TiO_(2))、Fe^(3+)-NTA和Ti^(4+)螯合在磷酸酯修饰的固相微球(Ti^(4+)-IMAC)3种不同富集方法磷酸化肽段的富集效率,并用不同分辨率的质谱仪评估富集稳定性。【结果】Ti^(4+)-IMAC富集效率最高,磷酸化位点数是TiO_(2)或Fe^(3+)-NTA方法的7倍以上;TiO_(2)和Fe^(3+)-NTA方法富集到的磷酸化位点数相差不大,与已报道的用TiO_(2)方法富集的磷酸化位点数目接近。Ti^(4+)-IMAC富集结果稳定性很好,高分辨率Lumos质谱仪鉴定到的磷酸化位点数是Velos的2.6倍。【结论】本研究较高效地实现了分枝菌酸小杆菌磷酸化事件的鉴定,共鉴定到2280个磷酸化蛋白、10880个磷酸化肽段及4433个可信磷酸化位点,有望用于其他微生物的磷酸化蛋白质组学研究。

[Objective]Mycolicibacterium smegmatis was used to explore more efficient strategies for enriching phosphorylated peptides in prokaryotes.[Methods]We evaluated the efficiency of three different methods,TiO_(2),Fe^(3+)NTA and Ti^(4+)-IMAC,for the enrichment of phosphopeptides.Further,we employed two mass spectrometers with different resolutions,Orbitrap Velos and Orbitrap Fusion Lumos,to assess the enrichment stability.[Results]Ti^(4+)-IMAC was the optimum enrichment method,with the number of phosphopeptides and sites enriched seven-fold that of TiO_(2) or Fe^(3+)-NTA.TiO_(2) and Fe^(3+)-NTA showed no significant difference in the number of phosphorylation sites enriched,which was similar to the results of the published works about TiO_(2).In addition,the detection results of two different mass spectrometers showed that Ti-IMAC enrichment was stable in two biological duplicate samples.The average phosphorylation sites detected by Lumos was 2.6-fold that by Velos.[Conclusion]Ti^(4+)-IMAC technique can efficiently accomplish high enrichment of phosphorylation events in M.smegmatis.We identified a total of 2280 phosphorylated proteins,10880 phosphorylated peptides and 4433 credible phosphorylation sites.Ti^(4+)-IMAC method can be widely used in the phosphoproteomics of othermicroorganisms.