应用核素标记相对和绝对定量技术结合纳升液-质联用筛选养血清脑颗粒对斑马鱼神经损伤预后的生物标志物  

Differential Proteomic Analysis of the Zebrafish Neuron Protective Effect of Cerebralcare Granules Using iTRAQ Labeling Coupled With 2DLC-MS/MS

作　　者：李晓稳[1] 佟玲[2] 李东翔[2] 李云飞[2] 孙国祥[1] 

机构地区：[1]沈阳药科大学药学院,沈阳110016 [2]创新中药关键技术国家重点实验室,天士力控股集团有限公司,天津300410

出　　处：《中国药学杂志》2016年第11期884-889,共6页Chinese Pharmaceutical Journal

基　　金：国家"重大新药创制"科技重大专项资助项目(2013ZX09402202)

摘　　要：目的分析斑马鱼神经元模型损伤给药治疗前后总蛋白差异,寻找养血清脑颗粒对斑马鱼神经元损伤预后的生物标记物及其作用机制。方法采用核素标记相对和绝对定量技术结合纳升液相色谱和串联质谱(Nano LC-MS/MS)对给药前后斑马鱼药效模型总蛋白进行分离并分析肽段,采用Proteinpilot 4.2软件对蛋白进行鉴定和定量分析,对获得的差异蛋白进行生物信息学分析,应用蛋白印迹法对可能的生物标记物进行验证。结果通过Nano LC-MS/MS分析鉴定出置信度>95%的蛋白质共1 933种,其中总差异表达蛋白质130种,模型组与模型给药组表达量上调≥1.5倍的蛋白质有9种,下调≤0.70倍的蛋白质有23种。通过GO分析显示,差异蛋白的分子功能主要集中在神经元离子通道的翻译和代谢过程;Western blot结果显示,与模型组相比,差异表达蛋白Ef1-α(elongation factor 1-alpha)和α-6-F(Na^+/K^+ transporting ATPase alpha 1 polypeptide)在给药后表达量下调,差异具有统计学意义(P<0.05),且与质谱结果一致。结论 iTRAQ联合Nano LC-MS/MS技术能高通量地筛选斑马鱼神经元损伤相关蛋白,初步阐述养血清脑颗粒对斑马鱼神经元损伤预后的作用机制。： OBJECTIVE To screen the differentially expressed proteins before and after administration of Cerebralcare granules to zebrafish central nerve injury （CNI） models and search practical markers and explore the molecular mechanism of the treatment. METHODS Isobaric tags for relative and absolute quantitation （iTRAQ） coupled with nano liquid chromatography-tandem mass spectrometry （Nano-LC-MS/MS） were used to analyze and identify differentially expressed serum proteins in the two groups. Bioinfor- matics was used to analyze the identified differentially expressed proteins, and the expression of representative differential proteins was verified by Western blotting. RESULTS With the high throughput proteomic technology of iTRAQ coupled with Nano-LC-MS/MS, 1 933 unique proteins were identified, and 130 proteins showed i〉 1.50 or 〈~ 0. 70 folds of changes during differentiation. The pro- teins detected in the zebrafish neuroendocrine brain had roles in the biological processes of translation, metabolic process and neuronal ion channel clustering. Efl-o~ （elongation factor 1-alpha） and ct-6-F （Na ＋/K ~ transporting ATPase alpha 1 polypeptide） were valida- ted by Western blotting. The two sets of data showed a statistically significant difference （ P 〈 0. 05 ）. These results were consistent with those from quantitative mass spectrometry. CONCLUSION A high-throughput screen for zebrafish central nerve injury proteins can be performed by a combined use of iTRAQ and Nano-LC-MS/MS, and the molecular mechanism of Cerebralcare granules treatment can thus be preliminarily explored.

关 键 词：养血清脑颗粒 蛋白组学 核素标记相对和绝对定量技术 斑马鱼神经元损伤模型 

分 类 号：R965[医药卫生—药理学]