Chen Emily I, McClatchy Daniel, Park Sung Kyu, Yates John R
Department of Chemical Physiology, The Scripps Research Institute, California 92037, USA.
Anal Chem. 2008 Nov 15;80(22):8694-701. doi: 10.1021/ac800606w. Epub 2008 Oct 21.
Methods for the global analysis of protein expression offer an approach to study the molecular basis of disease. Studies of protein expression in tissue, such as brain, are complicated by the need for efficient and unbiased digestion of proteins that permit identification of peptides by shotgun proteomic methods. In particular, identification and characterization of less abundant membrane proteins has been of great interest for studies of brain physiology, but often proteins of interest are of low abundance or exist in multiple isoforms. Parsing protein isoforms as a function of disease will be essential. In this study, we develop a digestion scheme using detergents compatible with mass spectrometry that improves membrane protein identification from brain tissue. We show the modified procedure yields close to 5,000 protein identifications from 1.8 mg of rat brain homogenate with an average of 25% protein sequence coverage. This procedure achieves a remarkable reduction in the amount of starting material required to observe a broad spectrum of membrane proteins. Among the proteins identified from a mammalian brain homogenate, 1897 (35%) proteins are annotated by Gene Ontology as membrane proteins, and 1225 (22.6%) proteins are predicted to contain at least one transmembrane domain. Membrane proteins identified included neurotransmitter receptors and ion channels implicated in important physiological functions and disease.
蛋白质表达的全局分析方法为研究疾病的分子基础提供了一种途径。在诸如大脑等组织中进行蛋白质表达研究时，由于需要对蛋白质进行高效且无偏倚的消化，以便通过鸟枪法蛋白质组学方法鉴定肽段，这使得研究变得复杂。特别是，对丰度较低的膜蛋白进行鉴定和表征一直是大脑生理学研究的热点，但感兴趣的蛋白质往往丰度较低或存在多种异构体。根据疾病情况解析蛋白质异构体至关重要。在本研究中，我们开发了一种使用与质谱兼容的去污剂的消化方案，该方案可提高从脑组织中鉴定膜蛋白的能力。我们表明，改进后的方法从1.8毫克大鼠脑匀浆中可鉴定出近5000种蛋白质，平均蛋白质序列覆盖率为25%。该方法显著减少了观察广泛膜蛋白所需的起始材料量。在从哺乳动物脑匀浆中鉴定出的蛋白质中，有1897种（35%）蛋白质经基因本体论注释为膜蛋白，1225种（22.6%）蛋白质预计至少含有一个跨膜结构域。鉴定出的膜蛋白包括与重要生理功能和疾病相关的神经递质受体和离子通道。
