双向电泳结合MDLC-MS/MS法分析阿替洛尔对映异构体作用后的血管平滑肌细胞差异表达蛋白

Proteomic analysis on differential protein expressions in VSMCs treated with atenolol enantiomers

目的以双向电泳结合多维色谱串联质谱策略,分析阿替洛尔对映异构体[(R)/(S)-ATN]作用于血管平滑肌细胞(vascular smooth muscle cells,VSMCs)后的差异表达蛋白质。方法以(R)/(S)-ATN分别作用于VSMCs,提取总蛋白质,双向电泳分离差异表达蛋白点,二维纳流液相色谱芯片(HPLC-CHIP,包含一支纳升级Zorbax 300SB-C18富集柱和一支纳升级Zorbax 300SB-C18分析柱)富集、分离差异表达蛋白酶解后的肽混合物,线性离子阱质谱进行在线分析,Spectrum Mill MS Proteomics Workbench自动分析MS和MS/MS数据,通过UniProt KB/SWISS-PORT数据库进行搜索。结果经比较、筛选,(S)-ATN干预VSMCs后的蛋白质组中有6个蛋白质点表达上调,分别是CrK相关底物蛋白、接头蛋白-3复合物b-1亚基、未定性蛋白C3orf19、线粒体苹果酸脱氢酶前体1、磷酸甘油醛脱氢酶、内皮细胞清道夫受体2前体。结论 (R)/(S)-ATN可引起广泛的蛋白质表达改变,除未定性蛋白C3orf19以外,其余5种蛋白质可能与细胞信号传导与跨膜转运、细胞能量代谢以及介导乙酰化低密度脂蛋白修饰与降解过程有关。

Objective Chiral medicines selectively interact with biological macromolecules through strict chiral recognition and demonstrate different pharmacokinetic characteristics and drug effects.To investigate the differential protein expressions in vascular smooth muscle cells（VSMCs） treated with 2 atenolol enantiomers,（R）-ATN and（S）-ATN.Methods Cell proliferation was detected with MTT assay when VSMCs were treated with（R）-ATN or（S）-ATN at 0,0.15,1.5,15,30,60,120,180 and 240 μmol/L respectively for 4 h.After the VSMCs were treated with 120 μmol/L（R）-ATN or（S）-ATN for 4 h,the cell proteins were extracted.Then,differential protein expression was assessed.The differential expression protein spots from（R）/（S）-ATN treated VSMCs were screened by 2-DE,and then analyzed by on-line two-dimensional nano liquid chromatography（HPLC-CHIP included an enrichment column of Zorbax 300SB-C18 and an analytical column of Zorbax 300SB C18） coupled with linear ion trap mass spectrometry.Results There were 6 differential proteins founded between VSMCs treated by（R）/（S）-ATN,including enhancer of filamentation 1,AP-3 complex subunit beta-1,malate dehydrogenase,mitochondrial precursor,glyceraldehyde-3-phosphate dehydrogenase,and uncharacterized protein C3orf19 were screened.The former 5 proteins were related to cardiovascular events.Conclusion Our results indicates that there are differential protein expressions in VSMCs treated with 2 atenolol enantiomers,（R）-ATN and（S）-ATN,so more attention should be paid chiral drugs.