Oligomers, fact or artefact? SDS-PAGE induces dimerization of β-amyloid in human brain samples
Oligomers, fact or artefact? SDS-PAGE induces dimerization of β-amyloid in human brain samples
复制标题
DOI：
10.1007/s00401-013-1083-z
复制
发表时间：
2013-04-01
影响因子：
12.7
通讯作者：
Barnham, Kevin J.
中科院分区：
文献类型：
--
作者：
Watt, Andrew D.;Perez, Keyla A.;Barnham, Kevin J.
脑内β-淀粉样蛋白（A β）的低级低聚物的形成被广泛认为是阿尔茨海默病（AD）发病机制的核心组成部分。然而，尽管在高通量和高分辨率的技术，如xMAP和质谱（MS）的进步，这些寡聚物的研究仍然依赖于低分辨率的蛋白质印迹和酶联免疫吸附测定。目前的研究使用十二烷基硫酸钠（SDS）聚丙烯酰胺凝胶电泳（PAGE）、xMAP和表面增强激光解吸/电离飞行时间MS比较了人类皮质组织中的A β谱，发现尽管关于单体A β水平的技术之间存在显著相关性，但只有SDS-PAGE能够检测A β的二聚体亚型。向AD组织中添加合成的双酪氨酸交联A β（1-40）Met（35）（O）证明，MS方法能够观察到飞摩尔浓度的二聚体A β，对单体A β水平无明显影响。焦点转向AD脑组织内SDS-PAGE和可观察到的二聚体A β水平之间的关联。这些研究表明，随着样品缓冲液中SDS浓度的增加，观察到二聚体A β水平增加。随后使用合成A β（1-42）证实了这一发现，并表明SDS诱导了二聚体A β的形成。SDS促进A β二聚化的发现对低级寡聚体在AD发病机制中的假定作用具有重要意义，并对寡聚A β作为治疗靶点的效用提出质疑。
The formation of low-order oligomers of beta-amyloid (A beta) within the brain is widely believed to be a central component of Alzheimer's disease (AD) pathogenesis. However, despite advances in high-throughput and high-resolution techniques such as xMAP and mass spectrometry (MS), investigations into these oligomeric species have remained reliant on low-resolution Western blots and enzyme-linked immunosorbent assays. The current investigation compared A beta profiles within human cortical tissue using sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis (PAGE), xMAP and surface enhanced laser desorption/ionization time-of-flight MS and found that whilst there was significant correlation across the techniques regarding levels of monomeric A beta, only SDS-PAGE was capable of detecting dimeric isoforms of A beta. The addition of synthetic di-tyrosine cross-linked A beta(1-40)Met(35)(O) to the AD tissue demonstrated that the MS methodology was capable of observing dimeric A beta at femto-molar concentrations, with no noticeable effect on monomeric A beta levels. Focus turned to the association between SDS-PAGE and levels of observable dimeric A beta within the AD brain tissue. These investigations revealed that increased levels of dimeric A beta were observed with increasing concentrations of SDS in the sample buffer. This finding was subsequently confirmed using synthetic A beta(1-42) and suggests that SDS was inducing the formation of dimeric A beta. The findings that SDS promotes A beta dimerization have significant implications for the putative role of low-order oligomers in AD pathogenesis and draw into question the utility of oligomeric A beta as a therapeutic target.