CAJ | 학술논문

目的利用二维液相色谱法分离小鼠肝脏磷酸化蛋白质组.方法取正常小鼠肝脏,裂解肝脏后利用磷酸盐金属亲和层析(PMAC)树脂提取磷酸化蛋白.将磷酸化蛋白用初始缓冲液置换后,进行一维色谱聚焦分离,再将一维收集的pH值在8.5至4.0之间的组分分别进行二维无孔硅胶反相高效液相色谱(RP-HPLC)分离.最后利用ProteoVue软件将二维UV图转换成胶图进行分析.结果成功提取了小鼠肝脏磷酸化蛋白,并在浓缩除盐后通过二维液相色谱分离成功建立小鼠肝脏磷酸化蛋白质组pI/UV图谱.其中,一维色谱聚焦分离pH值在8.5至4.0之间共收集16个组分,每个组分的二维UV图转换成pI/UV胶图.结论磷酸化蛋白纯化技术与二维液相色谱技术的结合是研究磷酸化蛋白质组的有效方法,为下一步鉴定和研究磷酸化蛋白的功能打下了基础.
목적이용이유액상색보법분리소서간장린산화단백질조.방법취정상소서간장,렬해간장후이용린산염금속친화층석(PMAC)수지제취린산화단백.장린산화단백용초시완충액치환후,진행일유색보취초분리,재장일유수집적pH치재8.5지4.0지간적조분분별진행이유무공규효반상고효액상색보(RP-HPLC)분리.최후이용ProteoVue연건장이유UV도전환성효도진행분석.결과성공제취료소서간장린산화단백,병재농축제염후통과이유액상색보분리성공건립소서간장린산화단백질조pI/UV도보.기중,일유색보취초분리pH치재8.5지4.0지간공수집16개조분,매개조분적이유UV도전환성pI/UV효도.결론린산화단백순화기술여이유액상색보기술적결합시연구린산화단백질조적유효방법,위하일보감정화연구린산화단백적공능타하료기출.
Objective To fractionate phosphoproteome of mouse liver by two-dimensional (2D) liquid phase chromatography fractionation. Methods Phosphoproteins were extracted from lysates of normal mice livers by phosphate metal affinity chromatography (PMAC) resin. The phosphoproteins were exchanged by start buffer and separated by chromatofocusing in the first dimension. Then the fractions between pH 8.5 and pH 4.0 were separated by non-porous silica (NPS) reverse-phase high performance liquid chromatography (RP-HPLC). Finally, the UV maps were converted into gel-like maps by ProteoVue software. Results Phosphoproteins of mouse liver were successfully extracted and fractionated by two dimensional liquid phase chromatographic fractionation after concentration and desalt. Then pI/UV map of mouse liver phosphoproteome was successfully set-up. There are 16 fractions between pH 8.5 and pH 4.0 after chromatofocusing in the first dimension and the UV maps of each fraction were converted into pI/UV gel-like maps. Conclusions Combination of technique of phosphoproteins enrichment and 2-D liquid phase chromatographic fractionation is an effective approach to research phosphoproteome and the key base for further identification and investigation of phosphoproteins.