王灼维, 彭福利, 王 媛, 童 维, 任 艳, 徐宁志, 刘斯奇＊

(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)

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.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)

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4mol/L尿素,20mmol/L三羟甲基氨基甲烷(Tris)-盐酸缓冲液(pH9.0)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(SDSPAGE)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。

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

Abstract:The analysis of membrane proteins is still a technical obstacle in p roteom ic investigation.A fundamental question is how to allow the hydrophobic proteins fully solubilizing in a p roper 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 in4mol/L urea,20mmol/L Tris-HCl buffer(pH9.0),and loaded onto a tandem Chromatography coup led with Q-Sepharose FF and Sephacryl S-200HR.with a linear NaCl gradient elution,the bound proteins were eluted and collected follow ed by sodium-dodecyl sulphate-polyacrylamide gel electrophoresis(SDSPAGE)to further separate the eluted proteins.The protein bound on SDS-PAGE were excised and in-gel digested by trypsin,while the digested pep tides were delivered to reversed-phase high performance liquid Chromatography(HPLC)and ion-trap mass spectrometry for the peptide identifications.O f a total of392proteins identified,306were membrane proteins or membrane associated proteins reported by literature.B ased on the calculation of hydrophobicity,the GRAVY(grand average of hydropathicity)scores of83p roteins are over or equal to0.00.Taking all the evidence,we have established an effective approach which is feasible in the investigation towards mouse liver membrane proteomics.

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

质膜是细胞与其生长环境相隔离的物理屏障[1]。……