基于Ti4+-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 MC2155 phosphoproteome based on the Ti4+-IMAC enrichment strategy

摘要:

【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(TiO₂)、Fe³⁺-NTA和Ti⁴⁺螯合在磷酸酯修饰的固相微球(Ti⁴⁺-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】Ti⁴⁺-IMAC富集效率最高，磷酸化位点数是TiO₂或Fe³⁺-NTA方法的7倍以上；TiO₂和Fe³⁺-NTA方法富集到的磷酸化位点数相差不大，与已报道的用TiO₂方法富集的磷酸化位点数目接近。Ti⁴⁺-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2 280个磷酸化蛋白、10 880个磷酸化肽段及4 433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。

Abstract:

[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,TiO₂,Fe³⁺-NTA and Ti⁴⁺-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] Ti⁴⁺-IMAC was the optimum enrichment method,with the number of phosphopeptides and sites enriched seven-fold that of TiO₂ or Fe³⁺-NTA.TiO₂ and Fe³⁺-NTA showed no significant difference in the number of phosphorylation sites enriched,which was similar to the results of the published works about TiO₂.In addition,the detection results of two different mass spectrometers showed that Ti⁴⁺-IMAC enrichment was stable in two biological duplicate samples.The average phosphorylation sites detected by Lumos was 2.6-fold that by Velos.[Conclusion]Ti⁴⁺-IMAC technique can efficiently accomplish high enrichment of phosphorylation events in M.smegmatis.We identified a total of 2 280 phosphorylated proteins,10 880 phosphorylated peptides and 4 433 credible phosphorylation sites.Ti⁴⁺-IMAC method can be widely used in the phosphoproteomics of other microorganisms.