二氧化钛富集磷酸肽方法优化及在腾冲嗜热厌氧菌磷酸化蛋白质组分析中的应用

Optimization of titanium dioxide enrichment of phosphopeptides and application in the Thermoanaerobacter tengcongensis phosphoproteome analysis

为了提高二氧化钛富集磷酸肽法对磷酸肽的富集效率,以6种标准蛋白酶切肽段混合物为研究对象,对二氧化钛富集磷酸肽过程中的乙腈比例、三氟乙酸比例、二氧化钛用量等条件分别进行了优化。结果表明在乙腈含量为80%(v/v),三氟乙酸含量为1%(v/v),二氧化钛用量与需要富集肽段的质量比为40∶1的条件下,可以取得较好的富集效果。将优化后的富集方法应用于腾冲嗜热厌氧菌磷酸化蛋白质的分析,初步鉴定到25个磷酸化蛋白质,为进一步研究这种极端环境下生存的低等生物的生命活动提供了参考信息。

Using titanium dioxide is a very good strategy for the phosphopeptide enrichment.There are many other factors can affect the enrichment efficiency,and the optimization of parameters was needed for better enrichment results.In this study,the peptide mixtures of six standard proteins were used as the model samples to evaluate and optimize the parameters such as the proportion of acetonitrile and trifluoroacetic acid in loading buffer and the TiO2-to-peptide ratio.The results showed that 80%（v/v） acetonitrile,1%（v/v） trifluoroacetic acid and 40∶1（m/m） TiO2-to-peptide ratio were the optimum parameters to obtain the best enrichment selectivity and maximum phosphopeptides identification.For the first time,the optimum enrichment conditions were applied for the phosphoproteome analysis of the Thermoanaerobacter tengcongensis,an anaerobic,saccharolytic,thermophilic bacterium isolated from a hot spring in Tengchong,China,and 25 phosphorylated proteins were identified in the preliminary experiment.The results provided a reference for further study on this organism survived under extreme environment.