斯达氏油脂酵母能在细胞内合成大量的油脂,其细胞内的脂类物质影响着蛋白质提取和后续的蛋白质组学分析.常见的一步蛋白质提取法很难从含有大量油脂的斯达氏酵母细胞中提取蛋白质,因此其蛋白质组学分析的结果较差.本文发展了顺序提取法提取斯达氏酵母中的蛋白质,并采用在线多维微柱反相液相色谱-串联质谱联用技术分析其蛋白质组;共鉴定到227个高可信度的蛋白质,其数目是一步提取法的两倍;此外,所鉴定到的参与基础代谢的蛋白质数目比一步提取法也显著增加.此方法可以有效提取含大量油脂的物种中的蛋白质,为油脂积累的分子机制研究提供重要的信息.%The oleaginous yeast Lipomyces starkeyi (L. starkeyi) is an excellent intracellular lipid producer. Thus, extraction of protein from lipid-rich L. starkeyi samples following conventional methods can be difficult, leading to poor data in terms of proteomic analysis. The presence of lipophilic components in those samples may also interfere with the extraction process and the downstream analysis. In this work, we developed a sequential extraction method for preparation and analysis of L. starkeyi proteome combining to an online multi- dimensional nano reversed-phase liquid chromatography-tandem mass spectrometry (μRPLC-MS/MS) strategy. Protein hits of high confidence reached 227 with false positive rate less than 0.1, twice of those identified from the one-buffer extraction preparation. Moreover, the protein hits related to primary metabolism was increased, which may be important to establish the molecular mechanism of lipid accumulation. The method should be valuable for protein extraction from oleaginous species.
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