多維離子交換色譜分離和串聯質譜鑒定在小鼠肝臟膜蛋白質組分析中的應用

王灼維, 彭福利, 王 媛, 童 維, 任 艷, 徐寧志, 劉斯奇＊

(1.中國科學院北京基因組研究所,北京101318;2.北京華大蛋白質研發中心有限公司,北京101318)

多維離子交換色譜分離和串聯質譜鑒定在小鼠肝臟膜蛋白質組分析中的應用

王灼維1,2#, 彭福利1#, 王 媛1,2, 童 維1,2, 任 艷1,2, 徐寧志2, 劉斯奇1,2＊

(1.中國科學院北京基因組研究所,北京101318;2.北京華大蛋白質研發中心有限公司,北京101318)

膜蛋白質在變性劑作用下能夠較充分地溶解。根據這一特點,我們試圖在變性劑溶液中采用串聯離子交換色譜法分離小鼠肝臟膜蛋白質。將小鼠肝臟膜蛋白質溶解于含有4mol/L尿素,20mmol/L三羥甲基氨基甲烷(Tris)-鹽酸緩沖液(pH9.0)中,用Q-Sepharose FF和Sephacryl S-200HR樹脂組成的色譜柱結合大部分溶解的膜蛋白質,然后采用氯化鈉線性梯度洗脫蛋白質,分步收集后采用十二烷基硫酸鈉-聚丙烯酰胺凝膠電泳(SDSPAGE)進一步分離洗脫組分的蛋白質。利用膠內胰蛋白酶消化技術將SDS-PAGE膠內分離的蛋白質降解為相應的肽段,然后以反相高效液相色譜分離和離子阱質譜儀鑒定肽段。根據文獻報道和蛋白質的功能分類,在所鑒定的392個蛋白質中有306個可能為膜蛋白質或膜結合蛋白質。蛋白質的疏水性計算表明,GRAVY(grand average of hydropathicity)得分大于或等于0.00的蛋白質有83個。綜上所述,我們有理由認為本實驗方法基本符合小鼠肝臟膜蛋白質組學研究的要求。

多維離子交換色譜;十二烷基硫酸鈉-聚丙烯酰胺凝膠電泳;反相高效液相色譜;串聯質譜;膜蛋白質;小鼠肝臟

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

質膜是細胞與其生長環境相隔離的物理屏障[1]。……