生物素标记法分析MCF-10A细胞质膜蛋白质

 目的 优化细胞膜蛋白分离纯化技术，以期实现快速、高效分析细胞膜蛋白质。方法 本文以乳腺细胞系MCF-10A为模型，用sulfo-NHS-LC-biotin标记完整的活细胞，使细胞表面蛋白生物素化，细胞裂解后利用链亲和素对膜蛋白进行分离富集，再洗脱膜蛋白，达到分离纯化的目的。利用液相层析偶合串联质谱技术（liquid chromatography-tandem mass spectrometry，LC-MS/MS）鉴定提取得到MCF 10A膜蛋白。结果 sulfo-NHS-LC-biotin浓度为0.2 mg/mL时得到的标记纯化膜蛋白最多，可作为标记最适浓度。利用这种方法鉴定出256个蛋白质，生物信息学分析表明其中膜蛋白占63%，比传统膜蛋白分离纯化方法得到的结果好。结论 这种方法省去常规分离膜蛋白时所需的超速离心，具有操作简单、可行性高、平行性高的特点，有望用于规模化肿瘤膜蛋白的差异比较，为肿瘤早期诊断及治疗提供新的靶标。

Analysis on plasma membrane proteins of MCF-10A by cell surface biotinylation

Objective To develop a rapid and efficient method for the analysis on plasma membrane proteins. Methods Mammary glandular cell line MCF-10A was chosen as the subject. After biotinylation of cell surface proteins in viable cells using sulfo-NHS-LC-biotin, cells were lysed in NP-40 lysis buffer. Biotinylated proteins were purified and enriched by streptavidin sepharose. Unbound proteins were removed by harsh washes, and than bound proteins were eluted. Then they were analyzed by LC-MS/MS. Results We were able to identify 256 kinds of proteins by this method, and up to 63% of all identified proteins were localized on the cell surface and plasma membrane as well as the classification of Gene Ontology database. Conclusions The method has been applied successfully to extract and identify membrane proteins, and it may be useful for comparative analysis of plasma membrane proteins in tumor detection and development of tumor therapeutics.