OLIGOMER FORMATION BY A-BETA PEPTIDES FOLLOWED BY AFM AND FTMS

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 淀粉样蛋白（A）肽可以形成淀粉样纤维和沉积物，这与阿尔茨海默病（AD）有关。已经提出二聚化与肽的毒性有关。一些研究已经表明，低聚物形成的早期阶段在导致淀粉样蛋白原纤维的过程中是至关重要的。一些因素如pH、金属离子和糖胺聚糖已被证明促进淀粉样纤维的形成。然而，关于在A？寡聚化的早期阶段中A？多聚体形成的详细机制仍然不清楚。可以形成复合物或与A？寡聚体相互作用的组分也可能影响原纤维形成过程。在本研究中， 将淀粉样蛋白肽1-40溶于去离子H2O、20 mM NH 4 HCO 3缓冲液和含1%甲酸的去离子H2O中，pH为2。还使A与交联剂BS 3（双[磺基琥珀酰亚胺基]辛二酸酯-d 0）在5 mM TEAB（三乙基碳酸氢铵）中以1：20的摩尔比反应。使用高分辨率MS和Bruker 12 T-SolariX-FTICR表征早期单体和低聚物，所述高分辨率MS配备有以正模式操作的纳米喷雾源。采用CID/ECD对单体、低聚物和加合物进行了串联质谱实验。用FTMS观察到A？寡聚体形成的早期阶段。中性pH下的样品在静置过夜后仅形成少量二聚体。A肽在pH 2缓冲液中在不到2小时内形成可观察到的二聚体、三聚体和四聚体，这与先前在低pH下加速A？原纤维形成的结果一致。ECD和CID片段化方法对于研究寡聚体和交联A？样品的结构是互补的。ECD对单体前体离子具有更好的裂解效率，而CID对二聚体的裂解效率更高。 CID和ECD片段的A？三聚体只产生单体和二聚体，表明低聚物松散结合。ECD在断裂松散结合的三聚体方面更有效。对于BS 3交联的A？，切割效率相对较低。在MS/MS实验中测定接头结合位点。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The Amyloid-¿ (A¿) peptide can form amyloid fibrils and deposits which have been linked to Alzheimer disease (AD). A¿ dimerization has been proposed to be related to the toxicity of the peptide. Several studies have already shown that the early stage of formation of the oligomers is critical in the process leading to the amyloid fibrils. Some factors such as pH, metal ions and glycosaminoglycans have been shown to promote formation of the amyloid fibrils. However, the detailed mechanisms regarding formation of the A¿ multimers in the early stages of A¿ oligomerization are still not clear. Components that can form a complex or interact with A¿ oligomers may affect the fibril formation process as well. In this study, Amyloid-¿ peptide 1-40 was dissolved in deionized H2O, 20 mM NH4HCO3 buffer and deionized H2O with 1% formic acid at pH2. A¿ was also reacted with cross-linker BS3 (bis[sulfosuccinimidyl] suberate-d0) at a molar ratio of 1:20 in 5 mM TEAB (triethylammonium bicarbonate). The early stage monomer and oligomers were characterized using a high resolution MS with the Bruker 12T-SolariX-FTICR, equipped with a nanospray source operated in positive mode. Tandem mass spectrometry experiments were performed on the monomer, oligomers and adducts by using CID/ECD. Early stages of oligomer formation of A¿ were observed with FTMS. A¿ sample at neutral pH, formed only a small amount of dimer after standing overnight. The A¿ peptide in pH2 buffer formed observable dimer, trimer and tetramer in less than 2 h, consistent with previous results of accelerated A¿ fibril formation at low pH. ECD and CID fragmentation method were complementary for studying the structure of the oligmer and crosslinked A¿ samples. ECD had better cleavage efficiency for monomer precursor ions while CID was more efficient in fragmenting the dimer. Both CID and ECD fragmentation of A¿ trimer yielded only monomer and dimer, indicating that the oligomers were loosely bonded. ECD was more efficient in breaking the loosely bonded trimer. For BS3 crosslinked A¿, the cleavage efficiency was relatively low. The linker binding sites were determined in the MS/MS experiments.