Söderberg Linda, Bogdanovic Nenad, Axelsson Birgitta, Winblad Bengt, Näslund Jan, Tjernberg Lars O
Karolinska Institutet and Dainippon Sumitomo Pharma Alzheimer Center (KASPAC), Neurotec, Novum.
Biochemistry. 2006 Aug 15;45(32):9849-56. doi: 10.1021/bi060331+.
Aggregation of the 40-42 residue amyloid beta-peptide (Abeta) into amyloid plaques is a central event in Alzheimer's disease (AD) pathogenesis. Many proteins have by immunohistochemical techniques been shown to codeposit with Abeta in AD plaques. It is possible that some of these could seed Abeta aggregation and therefore be found in the actual core of the plaque. Here, we present a highly sensitive method for unbiased biochemical analysis of plaque cores. A mild purification protocol based on centrifugation and filtration was used to purify intact plaque cores from human AD brain. The purified plaques were dispensed on a glass slide and viewed in a laser capture microscope, and plaque cores were catapulted into a tube cap by a laser beam. After dissolution in formic acid, plaques were digested and analyzed by liquid chromatography coupled online to electrospray/tandem mass spectrometry. One single plaque was found to be sufficient for positive identification of the main amyloid component. Remarkably, Abeta was the only protein identified when 200 plaques were isolated and analyzed with the present method. Thus, it is possible that no proteins copolymerize with Abeta in the plaque cores and that Abeta alone is sufficient for formation of plaque cores. In support of this notion, core-like structures were observed after incubation of synthetic Abeta for 2 weeks. We suggest that the method described here could be used for the general analysis of amyloid aggregates and inclusion bodies found in other neurodegenerative disorders and that plaque cores in AD brain are molecularly homogeneous structures.
由40 - 42个氨基酸残基组成的β淀粉样肽（Aβ）聚集成淀粉样斑块是阿尔茨海默病（AD）发病机制中的核心事件。通过免疫组织化学技术已表明，许多蛋白质与Aβ共沉积于AD斑块中。其中一些蛋白质有可能引发Aβ聚集，因此可能存在于斑块的实际核心部位。在此，我们提出一种用于对斑块核心进行无偏倚生化分析的高灵敏度方法。基于离心和过滤的温和纯化方案用于从人类AD大脑中纯化完整的斑块核心。将纯化后的斑块置于载玻片上，在激光捕获显微镜下观察，然后用激光束将斑块核心弹射到管帽中。在甲酸中溶解后，对斑块进行消化，并通过与电喷雾/串联质谱联用的液相色谱进行分析。发现仅一个斑块就足以阳性鉴定主要的淀粉样成分。值得注意的是，用本方法分离并分析200个斑块时，Aβ是唯一被鉴定出的蛋白质。因此，有可能在斑块核心中没有蛋白质与Aβ共聚，且仅Aβ自身就足以形成斑块核心。支持这一观点的是，合成Aβ孵育2周后观察到了类似核心的结构。我们认为，本文所述方法可用于对其他神经退行性疾病中发现的淀粉样聚集体和包涵体进行常规分析，并且AD大脑中的斑块核心是分子均一的结构。
