CAJ | 학술논문

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.
막단백질재변성제작용하능구교충분지용해.근거저일특점,아문시도재변성제용액중채용천련리자교환색보법분리소서간장막단백질.장소서간장막단백질용해우함유4 mol/L 뇨소,20 mmol/L 삼간갑기안기갑완(Tris)-염산완충액(pH 9.0)중,용Q-Sepharose FF화Sephacryl S-200HR수지조성적색보주결합대부분용해적막단백질,연후채용록화납선성제도세탈단백질,분보수집후채용십이완기류산납-취병희선알응효전영(SDS-PAGE)진일보분리세탈조분적단백질.이용효내이단백매소화기술장SDS-PAGE효내분리적단백질강해위상응적태단,연후이반상고효액상색보분리화리자정질보의감정태단.근거문헌보도화단백질적공능분류,재소감정적392개단백질중유306개가능위막단백질혹막결합단백질.단백질적소수성계산표명,GRAVY(grand average of hydropathicity)득분대우혹등우0.00적단백질유83개.종상소술,아문유이유인위본실험방법기본부합소서간장막단백질조학연구적요구.
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.