色谱

色谱 ›› 2011, Vol. 29 ›› Issue (05): 382-388.DOI: 10.3724/SP.J.1123.2011.00382

• 研究论文 • 上一篇    下一篇

基于蛋白顺序提取法分析斯达氏油脂酵母的蛋白质组

刘宏伟1, 赵鑫2, 程凯1, 赵宗宝2, 叶明亮1*   

  1. 1. 中国科学院大连化学物理研究所, 中国科学院分离分析化学重点实验室, 国家色谱研究分析中心, 辽宁 大连 116023; 2. 中国科学院大连化学物理研究所, 生物质高效转化课题组, 辽宁 大连 116023
  • 收稿日期:2011-03-19 修回日期:2011-04-22 出版日期:2011-05-28 发布日期:2011-05-28
  • 通讯作者: 叶明亮

Sequential extraction leading to improved proteomic analysis of the oleaginous yeast Lipomyces starkeyi

LIU Hongwei1, ZHAO Xin2, CHENG Kai1, ZHAO Zongbao2, YE Mingliang1*   

  1. State Key Laboratory of Marine Environmental Science, Environmental Science Research Center, College of Oceanography and Environmental Science, Xiamen University, Xiamen 361005, China
  • Received:2011-03-19 Revised:2011-04-22 Online:2011-05-28 Published:2011-05-28
  • Contact: YE Mingliang, PhD, Professor. Tel/Fax: (0411)84379620,
  • Supported by:
    National Natural Science Foundation of China (Nos. 20735004, 20675081)

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摘要： 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

关键词: 蛋白质组分析, 顺序提取, 斯达氏油脂酵母, 样品前处理, 在线多维微柱反相液相色谱-串联质谱

Abstract: 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 multidimensional 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.

Key words: proteome analysis, sample preparation, sequential extraction, Lipomyces starkeyi