中国药理学通报2012，Vol.28Issue(6)：881-884,4.DOI:10.3969/j.issn.1001-1978.2012.06.033
小鼠骨髓来源巨噬细胞的SILAC代谢标记及生物质谱分析
SILAC labeling of mouse bone marrow-derived macrophage and mass spectrometry
摘要
Abstract
Aim Mouse bone marrow-derived macrophages (BMM) are recognized as standard model cells of macrophages, thus serving as a key tool and objective cell type in pharmacology. Due to limited proliferation capacity of macrophages, meta-bolically labeling BMM remains as a challenge in macrophage biological investigations. Therefore, the focus of this study was to label BMM with stable isotope labeling with amino acids in cell culture (SILAC). Methods Methods include: mouse bone marrow isolation, 6-day differentiation with M-CSF to prepare BMM; meanwhile, to use SILAC to label global proteins, followed by cell lysis, SDS-PAGE separation, in-gel digestion, mass spectrometry and bioinformatics. Results On Day 6 post-differentiation, 96. 5% of total mouse bone marrow cells wereobserved to become mature BMM. 70 ku band in SDS-PAGE separation was adopted to test the labeling efficiencies. The number of heavy lysine labeled proteins were 18, 12 and 13 for the time points of Day 6, 8 10 post-differentiation. Eight proteins were repeatedly identified in all time points. Statistically, the labeling efficiencies of the three time points were (90. 62 ± 0. 03)% , (90. 23 ±0. 03)% and (90. 40 ± 0. 02)% , respectively. Conclusion These results ensure the feasibility of employing SILAC-based proteomics in macrophage-relevant pharmacological investigations.关键词
小鼠骨髓来源巨噬细胞(BMM)/SILAC/质谱/标记效率/蛋白质组/定量Key words
bone marrow-derived macrophage (BMM)/SILAC/ mass spectrometry/ labeling efficiency/ proteome/ quantification分类
医药卫生引用本文复制引用
王通,郭嘉慧,陈智鹏,银兴峰,马文心,崔毅峙..小鼠骨髓来源巨噬细胞的SILAC代谢标记及生物质谱分析[J].中国药理学通报,2012,28(6):881-884,4.基金项目
国家自然科学基金资助项目(No 81000516) （No 81000516）
国家重点基础研究发展计划(973计划)项目(No 2011CB910701) （973计划）
教育部博士点基金项目(No 20104401120008) （No 20104401120008）
暨南大学优秀本科推免研究生科研创新培育计划项目(No 50503590) （No 50503590）
