内毒素休克小鼠肝脏细胞质膜差异蛋白质组的二维液相色谱分析

Two-dimensional liquid chromatography analysis of differential proteome of liver plasma membrane in micewith endotoxic shock

目的:建立和优化肝脏细胞质膜蛋白质研究的方法系统,并对正常组小鼠和内毒素休克小鼠组肝脏细胞质膜蛋白质进行二维液相色谱分离,分析两者之间的差异表达谱.方法:BALB/c小鼠20只随机分为正常组和内毒素休克小鼠组(10只,LPS刺激3h,LPS剂量为35mg/kg),n=10.采用差速离心结合蔗糖密度梯度离心法分离和富集两组肝脏细胞质膜蛋白,分别将两组样品按蛋白质PI进行一维的色谱聚焦分离,然后每个PI组分再按蛋白质的疏水性经二维HPLC分离,利用ProteoVue软件将UV光吸收图谱转换成PI对疏水性的胶样图谱,再利用DeltaVue软件分析两组肝脏质膜蛋白质组的表达差异.结果:图像分析显示两组比值在≥2.0及≤0.5的差异蛋白点共有24个,与正常组比较,在内毒素休克小鼠的肝脏质膜蛋白中有16个蛋白点表达上调,8个蛋白点表达下调.结论:正常组和内毒素休克小鼠组的肝脏质膜蛋白质组有明显差异.

AIM： To establish and optimize the methods for plasma membrane proteome research, and comparatively analyze plasma membrane proteomes of normal control group and the group treated with LPS for 3 h. METHODS： Twenty BALB/c mice were assigned to 2 groups： the control group and LPS-treated group. Plasma membrane of liver cells from mice was extracted using differential centrifugation and sucrose density-gradient centrifugation. Plasma membrane proteins of two groups were fractionated according to pI using chromatofocusing with analytical columns in the first dimension, followed by separation of the proteins in each pI fraction using nonporous reversed-phase high performance liquid chromatography （HPLC）. A two-dimensional map of the protein content for each group based upon pI versus hydrophobicity as detected by UV absorption was generated and a differential display map indicating the presence of up- or down-regulated proteins was exhibited using ProteoVue and DeltaVue software. RESULTS： Image analysis of two-dimensional map revealed 24 differentially expressed proteins between the normal control group and that treated with LPS for 3 h. Compared with normal control, 16 protein fractions were up-regulated and 8 protein fractions were down-regulated in the group treated with LPS for 3 h .CONCLUSION： There is a significant difference of plasma membrane proteome between the normal control and the mice treated with LPS.