Characterization of α-Synuclein Multimer Stoichiometry in Complex Biological Samples by Electrophoresis.
Characterization of α-Synuclein Multimer Stoichiometry in Complex Biological Samples by Electrophoresis.
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DOI：
10.1021/acs.analchem.6b00419
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发表时间：
2016-04-05
影响因子：
7.4
通讯作者：
Moszczynska A
中科院分区：
文献类型：
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作者：
Killinger BA;Moszczynska A
大脑中α-突触核蛋白的异常聚集是帕金森病（PD）的标志。体内可溶性α-突触核蛋白以单体和几种多聚体形式存在，后者可能对α-突触核蛋白的生物学功能很重要。目前，缺乏可重复的方法来比较复杂生物样品之间的α-突触核蛋白多聚体丰度。在这里，我们开发了一种称为“多聚体PAGE”的方法，该方法将凝胶内化学交联与几种常见的电泳技术相结合，以测量脑组织裂解物中可溶性α-突触核蛋白多聚体的化学计量。结果表明，大鼠脑中可溶性α-突触核蛋白以几种约56 kDa（αS56）、80 kDa（αS80）和100 kDa（αS100）的高分子量物质存在，这些物质在蓝色非变性凝胶电泳（BN-PAGE）过程中与内源性脂质、去污剂和/或胶束共迁移。内源性脂质与α-突触核蛋白的共提取对于可溶性α-突触核蛋白多聚体的检测是必需的。脑组织在小缓冲液体积（>50 mg组织/1 mL缓冲液）中的均质化增加了相对脂质提取，并随后通过多聚体-PAGE产生丰富的可溶性多聚体检测。通过直接交联可溶性裂解物捕获的α-突触核蛋白多聚体类似于在多聚体-PAGE后观察到的那些。多聚体（αS80）与单体（αS17）的比例随多聚体-PAGE中蛋白质输入量的增加而线性增加，表明在一定程度上，在电泳过程中也形成了多聚体。总的来说，可溶性α-突触核蛋白在组织破坏后维持脂质相互作用，并且当这种脂质-蛋白质复合物被保留时容易形成多聚体。一旦多聚体PAGE技术得到验证，相对化学计量比较可以同时进行14个生物样品之间。多聚体聚丙烯酰胺凝胶电泳为研究影响α-突触核蛋白多聚化的分子因素提供了一种简单廉价的生化技术。
The aberrant aggregation of α-synuclein in the brain is a hallmark of Parkinson’s disease (PD). In vivo soluble α-synuclein occurs as a monomer and several multimers, the latter of which may be important for the biological function of α-synuclein. Currently, there is a lack of reproducible methods to compare α-synuclein multimer abundance between complex biological samples. Here we developed a method, termed “multimer-PAGE,” that combines in-gel chemical cross-linking with several common electrophoretic techniques to measure the stoichiometry of soluble α-synuclein multimers in brain tissue lysates. Results show that soluble α-synuclein from the rat brain exists as several high molecular weight species of approximately 56 kDa (αS56), 80 kDa (αS80), and 100 kDa (αS100) that comigrate with endogenous lipids, detergents, and/or micelles during blue native gel electrophoresis (BN-PAGE). Co-extraction of endogenous lipids with α-synuclein was essential for the detection of soluble α-synuclein multimers. Homogenization of brain tissue in small buffer volumes (>50 mg tissue per 1 mL buffer) increased relative lipid extraction and subsequently resulted in abundant soluble multimer detection via multimer-PAGE. α-Synuclein multimers captured by directly cross-linking soluble lysates resembled those observed following multimer-PAGE. The ratio of multimer (αS80) to monomer (αS17) increased linearly with protein input into multimer-PAGE, suggesting to some extent, multimers were also formed during electrophoresis. Overall, soluble α-synuclein maintains lipid interactions following tissue disruption and readily forms multimers when this lipid–protein complex is preserved. Once the multimer-PAGE technique was validated, relative stoichiometric comparisons could be conducted simultaneously between 14 biological samples. Multimer-PAGE provides a simple inexpensive biochemical technique to study the molecular factors influencing α-synuclein multimerization.