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

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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.