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.
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.
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