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目的利用双向电泳和质谱技术对小鼠骨髓来源未成熟树突状细胞(dendritic cells,DC)表达的蛋白质进行分析。方法 IL-4和GM-CSF诱导未成熟DC的分化,提取细胞总蛋白,定量。双向凝胶电泳分离蛋白质组分,胶体银染显色,PDQuest进行图像分析后选取蛋白点,胶内酶解后MALDI-TOF MS进行肽指纹图谱鉴定,MS-Fit分析质谱数据。结果细胞生长情况和表面标志物检测表明获得了高纯度的未成熟DC。图像分析结果显示,DC中检测到了(660±10)个蛋白点,主要分布在相对分子质量28×103～100×103,等电点5.0～9.0之间。鉴定了87个蛋白点,其中与未成熟DC免疫调节功能相关的蛋白质有40个(45.98%);与大分子代谢相关的有37个(42.53%)。结论成功分离到了约660个小鼠骨髓来源未成熟DC表达的蛋白质,鉴定出了其中87个蛋白。
Objective To analyze the protein expression of mouse bone marrow-derived immature dendritic cells (DCs) by two-dimensional electrophoresis and mass spectrometry. Methods IL-4 and GM-CSF induced differentiation of immature DCs, total cellular protein was extracted and quantitated. Two-dimensional gel electrophoresis (SDS-PAGE) was used to separate the protein components, colloidal silver staining, PDQuest image analysis, protein spots selection, gel digestion MALDI-TOF MS peptide fingerprinting identification, MS-Fit mass spectrometry data. Results Cell growth and surface marker assays indicated that high purity immature DCs were obtained. The results of image analysis showed that (660 ± 10) protein spots were detected in DC, which mainly distributed in the relative molecular mass of 28 × 103 ~ 100 × 103 and the isoelectric point of 5.0 ~ 9.0. 87 protein spots were identified, of which 40 (45.98%) were related to the immunomodulatory function of immature DC and 37 (42.53%) were related to macromolecular metabolism. Conclusion The protein of immature DCs from about 660 mouse bone marrow was successfully isolated and 87 proteins were identified.