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

目的作为模型细胞之一,小鼠骨髓来源巨噬细胞(BMM)是药理学、药效学研究的重要工具和对象。然而,由于巨噬细胞增殖能力较弱,代谢标记细胞内蛋白一直是巨噬细胞生物学中的一个难题。因此,本研究以SILAC(stable i-sotope labeling with amino acids in cell culture)方法标记BMM。方法小鼠骨髓细胞的分离,以M-CSF诱导6 d并制备BMM,同时,SILAC标记细胞内蛋白;进而,裂解细胞并收集蛋白质,以SDS-PAGE进行初步分离,经胶内酶解成肽段,再通过质谱分析测定,统计得其标记效率。结果小鼠骨髓细胞在分化6 d时点成熟BMM可占细胞总数的0.965。以70 ku条带为研究对象,d 6、8和d 10鉴定到重链赖氨酸标记蛋白数分别为18、12和13个。其中,有8个蛋白在该3个时点中均被检出。统计结果表明3个时点的重链赖氨酸蛋白标记效率分别为(90.62±0.03)%、(90.23±0.03)%和(90.40±0.02)%,达到了SILAC研究的要求。结论该方法解决了BMM的SILAC标记问题,可为以巨噬细胞为研究对象的药理学研究提供有效的研究手段。

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,metabolically 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 were observed 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.