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首页> 美国卫生研究院文献>Molecular Cellular Proteomics : MCP >Biochemical Fractionation and Stable Isotope Dilution Liquid Chromatography-mass Spectrometry for Targeted and Microdomain-specific Protein Quantification in Human Postmortem Brain Tissue
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Biochemical Fractionation and Stable Isotope Dilution Liquid Chromatography-mass Spectrometry for Targeted and Microdomain-specific Protein Quantification in Human Postmortem Brain Tissue

机译：生化分离和稳定同位素稀释液相色谱-质谱法用于人类死后脑组织中靶向和微域特异性蛋白质定量

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Synaptic architecture and its adaptive changes require numerous molecular events that are both highly ordered and complex. A majority of neuropsychiatric illnesses are complex trait disorders, in which multiple etiologic factors converge at the synapse via many signaling pathways. Investigating the protein composition of synaptic microdomains from human patient brain tissues will yield valuable insights into the interactions of risk genes in many disorders. These types of studies in postmortem tissues have been limited by the lack of proper study paradigms. Thus, it is necessary not only to develop strategies to quantify protein and post-translational modifications at the synapse, but also to rigorously validate them for use in postmortem human brain tissues. In this study we describe the development of a liquid chromatography-selected reaction monitoring method, using a stable isotope-labeled neuronal proteome standard prepared from the brain tissue of a stable isotope-labeled mouse, for the multiplexed quantification of target synaptic proteins in mammalian samples. Additionally, we report the use of this method to validate a biochemical approach for the preparation of synaptic microdomain enrichments from human postmortem prefrontal cortex. Our data demonstrate that a targeted mass spectrometry approach with a true neuronal proteome standard facilitates accurate and precise quantification of over 100 synaptic proteins in mammalian samples, with the potential to quantify over 1000 proteins. Using this method, we found that protein enrichments in subcellular fractions prepared from human postmortem brain tissue were strikingly similar to those prepared from fresh mouse brain tissue. These findings demonstrate that biochemical fractionation methods paired with targeted proteomic strategies can be used in human brain tissues, with important implications for the study of neuropsychiatric disease.
机译：突触结构及其适应性变化需要大量高度有序且复杂的分子事件。大多数神经精神疾病是复杂的性状疾病，其中多种病因通过多种信号途径汇聚在突触处。研究来自人类患者脑组织的突触微区的蛋白质组成将对许多疾病中的风险基因的相互作用产生有价值的见解。死后组织中的这类研究由于缺乏适当的研究范例而受到限制。因此，不仅有必要开发策略来量化突触中的蛋白质和翻译后修饰，而且有必要严格验证它们是否可用于死后人类脑组织。在这项研究中，我们描述了一种液相色谱选择的反应监测方法的开发，该方法使用了从稳定同位素标记的小鼠的脑组织中制备的稳定同位素标记的神经元蛋白质组标准品，用于哺乳动物样品中目标突触蛋白的多重定量。此外，我们报告了这种方法的使用，以验证一种生物化学方法，用于从人死后前额叶皮层制备突触微区富集。我们的数据表明，采用真正的神经元蛋白质组标准物的靶向质谱分析方法有助于对哺乳动物样品中的100多种突触蛋白进行准确而精确的定量，并有可能定量1000多种蛋白。使用这种方法，我们发现从人类死后脑组织制备的亚细胞部分中的蛋白质富集与从新鲜小鼠脑组织制备的蛋白质中的富集惊人地相似。这些发现表明，生化分离方法与靶向蛋白质组学策略相结合可用于人脑组织，对神经精神疾病的研究具有重要意义。

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