小鼠肝脏磷酸化蛋白质组的二维液相色谱分离

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,裂解肝脏后利用金属磷酸盐亲和层析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坚实的基础。

Two-dimensional liquid chromatographic fractionation of mouse liver phosphoproteome

Objective To fractionate mouse liver phosphoproteome by two-dimensional liquid chromatography. Methods The phosphoproteins were extracted from the lysates of normal mouse livers with phosphate metal affinity chromatography resin. The phosphoproteins were exchanged by the initial buffer and separated by chromatofocusing in the first dimension. The fractions with pH value between 8.5 and 4.0 were separated by non-porous silica reverse-phase high-performance liquid chromatography. Finally, the UV maps were converted into gel maps by ProteoVue software. Results The phosphoproteins of mouse liver were successfully extracted and fractionated by two-dimensional liquid chromatographic fractionation after concentration and desalting. The pI/UV map of mouse liver phospho-proteomic was obtained successfully. There were 16 fractions with pH between 8.5 and 4.0 after chromatofocusing in the first dimension and the UV maps of each fraction were converted into pI/UV gel maps. Conclusion Combination of phosphoprotein enrichment and two-dimensional liquid chromatographic fractionation is an effective approach of phosphoproteomic studies.