Abstract
Formation and accumulation of fibrillar plaques and aggregates of ß-amyloid peptide (Aß) in brain have been recognized as characteristics of Alzheimer’s disease (AD). Oligomeric aggregates of Aß are considered critical intermediates leading to progressive neurodegeneration; however, molecular details of the oligomerization and aggregation pathway and the structures of Aß-oligomers are hitherto unclear. Using an in vitro fibril formation procedure of Aß(1–40), ß-amyloid aggregates were prepared and insoluble aggregates separated from soluble products by centrifugation. In this study, ion mobility mass spectrometry (IM-MS) was applied in combination with electron paramagnetic resonance spectroscopy (EPR) to the identification of the components of Aß-oligomers, and to their structural and topographical characterization. The formation of Aß-oligomers and aggregates was monitored by gel electrophoresis, and Aß-oligomer bands were identified by in-gel tryptic digestion and matrix-assisted laser desorption ionization–mass spectrometry (MALDI-MS) to consist predominantly of Aß(1–40) peptide. First, ion mobility-MS studies of soluble Aß-aggregates prepared by incubation for 5 days were performed on a quadrupole time-of-flight mass spectrometer and revealed (1) the presence of at least two different conformational states, and (2), the formation of Met-35 oxidized products. For estimation of the size of Aß-aggregates using EPR spectroscopy, a modified Aß(1–40) peptide containing an additional N-terminal cysteine residue was prepared, and a 3-(2-iodoacetamido)-2,2,5,5-tetramethyl-1-pyrrolidinyloxy radical spin label derivative (IPSL) was coupled by S-alkylation. The EPR spectra of the spin-labeled Cys-Aß(1–40) oligomers were matched with spectra simulations using a multi-component simulation strategy, resulting in complete agreement with the gel electrophoresis results.
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Acknowledgments
We thank Marilena Manea and Marcel Leist for expert help with the synthesis of spin-labeled Aß-peptide derivatives, and critical discussion of the manuscript, and Martin Spitzbarth for help with the EPR simulations. This work was supported by the International Research Center “Proteostasis” at the University of Konstanz, and by the Deutsche Forschungsgemeinschaft, Bonn, Germany (DR 743/2-1).
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Iuraşcu, M.I., Cozma, C., Tomczyk, N. et al. Structural characterization of ß-amyloid oligomer-aggregates by ion mobility mass spectrometry and electron spin resonance spectroscopy. Anal Bioanal Chem 395, 2509–2519 (2009). https://doi.org/10.1007/s00216-009-3164-3
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DOI: https://doi.org/10.1007/s00216-009-3164-3