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

李晓稳, 佟玲, 李东翔, 李云飞, 孙国祥

中国药学杂志 ›› 2016, Vol. 51 ›› Issue (11) : 884-889.

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中国药学杂志 ›› 2016, Vol. 51 ›› Issue (11) : 884-889. DOI: 10.11669/cpj.2016.11.005
论著

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

  • 李晓稳1, 佟玲2, 李东翔2, 李云飞2, 孙国祥1*
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Differential Proteomic Analysis of the Zebrafish Neuron Protective Effect of Cerebralcare Granules Using iTRAQ Labeling Coupled With 2DLC-MS/MS

  • LI Xiao-wen1, TONG Ling2, LI Dong-xiang2, LI Yun-fei2, SUN Guo-xiang1*
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文章历史 +

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。

Abstract

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. Bioinformatics 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 ≥ 1.50 or ≤ 0.70 folds of changes during differentiation. The proteins detected in the zebrafish neuroendocrine brain had roles in the biological processes of translation, metabolic process and neuronal ion channel clustering. Ef1-α (elongation factor 1-alpha) and α-6-F (Na+/K+ transporting ATPase alpha 1 polypeptide) were validated 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

Key words

Cerebralcare granule / proteomic / isobaric tags for relative and absolute quantitation / zebrafish central nerve injury

引用本文

导出引用
李晓稳, 佟玲, 李东翔, 李云飞, 孙国祥. 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[J]. 中国药学杂志, 2016, 51(11): 884-889 https://doi.org/10.11669/cpj.2016.11.005
LI Xiao-wen, TONG Ling, LI Dong-xiang, LI Yun-fei, SUN Guo-xiang. Differential Proteomic Analysis of the Zebrafish Neuron Protective Effect of Cerebralcare Granules Using iTRAQ Labeling Coupled With 2DLC-MS/MS[J]. Chinese Pharmaceutical Journal, 2016, 51(11): 884-889 https://doi.org/10.11669/cpj.2016.11.005
中图分类号: R965   

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基金

国家“重大新药创制”科技重大专项资助项目(2013ZX09402202)
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