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
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国家自然科学基金(32141003,31901037);京津冀基础研究合作专项(J200001)


In-depth characterization of Mycolicibacterium smegmatis MC2155 phosphoproteome based on the Ti4+-IMAC enrichment strategy
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  • MENG Shuhong

    MENG Shuhong

    College of Life Sciences, Hebei University, Baoding 071002, Hebei, China;State Key Laboratory of Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences Beijing, Research Unit of Proteomics & Research and Development of New Drug of Chinese Academy of Medical Sciences, Institute of Lifeomics, Academy of Military Medical Sciences of Academy of Military Science, Beijing 102206, China
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  • CHANG Lei

    CHANG Lei

    State Key Laboratory of Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences Beijing, Research Unit of Proteomics & Research and Development of New Drug of Chinese Academy of Medical Sciences, Institute of Lifeomics, Academy of Military Medical Sciences of Academy of Military Science, Beijing 102206, China
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  • LIU Fengsong

    LIU Fengsong

    College of Life Sciences, Hebei University, Baoding 071002, Hebei, China
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  • XU Ping

    XU Ping

    State Key Laboratory of Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences Beijing, Research Unit of Proteomics & Research and Development of New Drug of Chinese Academy of Medical Sciences, Institute of Lifeomics, Academy of Military Medical Sciences of Academy of Military Science, Beijing 102206, China
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  • ZHANG Yao

    ZHANG Yao

    State Key Laboratory of Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences Beijing, Research Unit of Proteomics & Research and Development of New Drug of Chinese Academy of Medical Sciences, Institute of Lifeomics, Academy of Military Medical Sciences of Academy of Military Science, Beijing 102206, China
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    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.

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蒙书红,常蕾,柳峰松,徐平,张瑶. 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[J]. 微生物学报, 2022, 62(10): 3768-3783

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  • 收稿日期:2022-02-08
  • 最后修改日期:2022-06-22
  • 在线发布日期: 2022-09-24
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