色谱

色谱 ›› 2010, Vol. 28 ›› Issue (2): 115-122.

• 特别策划 • 上一篇    下一篇

多维离子交换色谱分离和串联质谱鉴定在小鼠肝脏膜蛋白质组分析中的应用

王灼维1,2#, 彭福利1#, 王媛1,2, 童维1,2, 任艳1,2, 徐宁志2, 刘斯奇1,2*   

  1. 1. 中国科学院北京基因组研究所, 北京 101318; 2. 北京华大蛋白质研发中心有限公司, 北京 101318
  • 收稿日期:2009-12-18 修回日期:2010-02-01 出版日期:2010-02-28 发布日期:1981-06-25
  • 通讯作者: 刘斯奇

Analysis of mouse liver membrane proteins using multidimensional ion exchange chromatography and tandem mass spectrometry

WANG Zhuowei1,2#, PENG Fuli1#, WANG Yuan1,2, TONG Wei1,2, REN Yan1,2, XU Ningzhi2, LIU Siqi1,2*   

  1. 1. Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 101318, China; 2. Beijing Protein Innovation, Beijing 101318, China
  • Received:2009-12-18 Revised:2010-02-01 Online:2010-02-28 Published:1981-06-25
  • Contact: LIU Siqi

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2

摘要： 膜蛋白质在变性剂作用下能够较充分地溶解。根据这一特点,我们试图在变性剂溶液中采用串联离子交换色谱法分离小鼠肝脏膜蛋白质。将小鼠肝脏膜蛋白质溶解于含有4 mol/L尿素,20 mmol/L三羟甲基氨基甲烷(Tris)-盐酸缓冲液(pH 9.0)中,用Q-Sepharose FF和Sephacryl S-200HR树脂组成的色谱柱结合大部分溶解的膜蛋白质,然后采用氯化钠线性梯度洗脱蛋白质,分步收集后采用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)进一步分离洗脱组分的蛋白质。利用胶内胰蛋白酶消化技术将SDS-PAGE胶内分离的蛋白质降解为相应的肽段,然后以反相高效液相色谱分离和离子阱质谱仪鉴定肽段。根据文献报道和蛋白质的功能分类,在所鉴定的392个蛋白质中有306个可能为膜蛋白质或膜结合蛋白质。蛋白质的疏水性计算表明,GRAVY(grand average of hydropathicity)得分大于或等于0.00的蛋白质有83个。综上所述,我们有理由认为本实验方法基本符合小鼠肝脏膜蛋白质组学研究的要求。

关键词: 串联质谱, 多维离子交换色谱, 反相高效液相色谱, 膜蛋白质, 十二烷基硫酸钠-聚丙烯酰胺凝胶电泳, 小鼠肝脏

Abstract: The analysis of membrane proteins is still a technical obstacle in proteomic investigation. A fundamental question is how to allow the hydrophobic proteins fully solubilizing in a proper solvent environment. We propose that the denatured membrane proteins in high denaturant solution are fully ionized and separated through ion exchange chromatography. The membrane proteins prepared from a mouse liver were dissolved in 4 mol/L urea, 20 mmol/L Tris-HCl buffer (pH 9.0), and loaded onto a tandem chromatography coupled with Q-Sepharose FF and Sephacryl S-200HR. With a linear NaCl gradient elution, the bound proteins were eluted and collected followed by sodium-dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) to further separate the eluted proteins. The protein bound on SDS-PAGE were excised and in-gel digested by trypsin, while the digested peptides were delivered to reversed-phase high performance liquid chromatography (HPLC) and ion-trap mass spectrometry for the peptide identifications. Of a total of 392 proteins identified, 306 were membrane proteins or membrane associated proteins reported by literature. Based on the calculation of hydrophobicity, the GRAVY (grand average of hydropathicity) scores of 83 proteins are over or equal to 0.00. Taking all the evidence, we have established an effective approach which is feasible in the investigation towards mouse liver membrane proteomics.

Key words: membrane protein, mouse liver, multidimensional ion-exchange chromatography, reversed-phase high performance liquid chromatography (RP-HPLC), sodium-dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), tandem mass spectrometry (MS/MS)