Department of Neurology, Knight Alzheimer's Disease Research Center, Hope Center for Neurological Disorders, Washington University School of Medicine, St. Louis, MO 63110, USA.
Anal Biochem. 2013 Sep 1;440(1):56-62. doi: 10.1016/j.ab.2013.04.031. Epub 2013 May 25.
Abundant evidence suggests a central role for the amyloid-beta (Aβ) peptide in Alzheimer's disease (AD) pathogenesis. Production and clearance of different Aβ isoforms have been established as targets of proposed disease-modifying therapeutic treatments of AD. However, previous studies used multiple sequential purification steps to isolate the isoforms individually and quantitate them based on a common mid-domain peptide. We created a method to simultaneously purify Aβ isoforms and quantitate them by the specific C-terminal peptides in order to investigate Aβ isoform physiology in the central nervous system. By using standards generated from in vitro metabolic labeling, the relative quantitation of four peptides representing total amount of Aβ (Aβ-Total), Aβ38, Aβ40, and Aβ42 were achieved both in cell culture and in human cerebrospinal fluid (CSF). Standard curves for each isoform demonstrated good sensitivity with very low limits of detection and high accuracy. Because the assay does not require antibody development for each Aβ isoform peptide, significant improvements in the throughput and accuracy of isoform quantitation were achieved.
大量证据表明，β淀粉样肽（Aβ）在阿尔茨海默病（AD）发病机制中起核心作用。不同 Aβ 亚型的产生和清除已被确定为 AD 潜在治疗方法的目标。然而，之前的研究使用了多个连续的纯化步骤来单独分离亚型，并基于共同的中间域肽对其进行定量。我们创建了一种方法，通过特异性 C 末端肽同时纯化 Aβ 亚型并对其进行定量，以便研究中枢神经系统中 Aβ 亚型的生理学。通过使用体外代谢标记产生的标准品，我们在细胞培养和人脑脊液（CSF）中实现了代表 Aβ 总量（Aβ-Total）、Aβ38、Aβ40 和 Aβ42 的四种肽的相对定量。每个亚型的标准曲线均表现出良好的灵敏度，检测限非常低，准确性高。由于该测定法不需要针对每个 Aβ 亚型肽开发抗体，因此在亚型定量的通量和准确性方面取得了显著提高。
