Structural characterization of ß-amyloid oligomer-aggregates by ion mobility mass spectrometry and electron spin resonance spectroscopy

Abstract

Formation and accumulation of fibrillar plaques and aggregates of beta-amyloid peptide (Abeta) 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 Abeta-oligomers are hitherto unclear. Using an in vitro fibril formation procedure of Abeta(1-40), beta-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 Abeta-oligomers, and to their structural and topographical characterization. The formation of Abeta-oligomers and aggregates was monitored by gel electrophoresis, and Abeta-oligomer bands were identified by in-gel tryptic digestion and matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) to consist predominantly of Abeta(1-40) peptide. First, ion mobility-MS studies of soluble Abeta-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 Abeta-aggregates using EPR spectroscopy, a modified Abeta(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-Abeta(1-40) oligomers were matched with spectra simulations using a multi-component simulation strategy, resulting in complete agreement with the gel electrophoresis results.