Adams Daniel J, Nemkov Travis G, Mayer John P, Old William M, Stowell Michael H B
The Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, Colorado, United States of America.
The Department of Biochemistry and Molecular Genetics, University of Colorado Anschutz Medical Campus, Denver, Colorado, United States of America.
PLoS One. 2017 Aug 9;12(8):e0182804. doi: 10.1371/journal.pone.0182804. eCollection 2017.
Understanding the pathophysiology of Alzheimer disease has relied upon the use of amyloid peptides from a variety of sources, but most predominantly synthetic peptides produced using t-butyloxycarbonyl (Boc) or 9-fluorenylmethoxycarbonyl (Fmoc) chemistry. These synthetic methods can lead to minor impurities which can have profound effects on the biological activity of amyloid peptides. Here we used a combination of cytotoxicity assays, fibrillation assays and high resolution mass spectrometry (MS) to identify impurities in synthetic amyloid preparations that inhibit both cytotoxicity and aggregation. We identify the Aβ42Δ39 species as the major peptide contaminant responsible for limiting both cytotoxicity and fibrillation of the amyloid peptide. In addition, we demonstrate that the presence of this minor impurity inhibits the formation of a stable Aβ42 dimer observable by MS in very pure peptide samples. These results highlight the critical importance of purity and provenance of amyloid peptides in Alzheimer's research in particular, and biological research in general.
对阿尔茨海默病病理生理学的理解依赖于使用来自多种来源的淀粉样肽，但最主要的是使用叔丁氧羰基（Boc）或芴甲氧羰基（Fmoc）化学方法生产的合成肽。这些合成方法可能会导致微量杂质，而这些杂质可能会对淀粉样肽的生物活性产生深远影响。在这里，我们结合细胞毒性测定、纤维化测定和高分辨率质谱（MS）来鉴定合成淀粉样制剂中抑制细胞毒性和聚集的杂质。我们确定Aβ42Δ39物种是限制淀粉样肽细胞毒性和纤维化的主要肽污染物。此外，我们证明这种微量杂质的存在会抑制在非常纯的肽样品中通过质谱观察到的稳定Aβ42二聚体的形成。这些结果突出了淀粉样肽的纯度和来源在阿尔茨海默病研究乃至一般生物学研究中的至关重要性。
