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.

Unraveling the Early Events of Amyloid-β Protein (Aβ) Aggregation: Techniques for the Determination of Aβ Aggregate Size

“…Since Aβ aggregation is a process that involves changes in protein conformation, native-PAGE is often a suitable technique to detect various sizes of Aβ species. A study by Iurascu et al used both SDS-PAGE and Tris-tricine PAGE to analyze the species formed by a solution of Aβ 1-40 solubilized in fibril growth buffer at pH 7.5 for 5 days at 37 °C [48]. They found that SDS-PAGE was able to detect Aβ 1-40 monomeric species, Aβ 1-40 oligomeric species of 20 kDa, and high molecular weight aggregates >98 kDa.…”

“…Iurascu et al . utilized SDS-PAGE in combination with MALDI-MS to analyze a solution of Aβ 1-40 solubilized in fibril growth buffer at pH 7.5 for 5 days at 37 °C [48]. MALDI-MS indicated that the soluble fraction contained two different ion mobilities, indicative of oligomerization.…”

CuII Binding to Various Forms of Amyloid‐β Peptides: Are They Friends or Foes?

“…[65] These methods include recognition by specific anti-bodies, [66] mass spectrometry, [45] fluorescence resonance energy transfer (FRET), [58] and electrophoresis on native gel. [67] Furthermore, the morphology of the formed aggregates can be evaluated by means of microscopy (TEM or AFM). [49,68] Each of these techniques offers advantages but also suffers from drawbacks in terms of technique-related artifacts.…”

Monitoring Insulin Aggregation via Capillary Electrophoresis

“…Various techniques have been utilized to detect soluble and low-molecular weight oligomeric species formed by amyloid proteins such as atomic force microscopy (AFM) [12,14], light scattering [12,14], hydrogen-deuterium exchange mass spectrometry [15,16], matrix assisted laser desorption ionization mass spectrometry (MALDI-MS) [17,18], electrospray ionization mass spectrometry (ESI-MS) [19], ion mobility mass spectrometry (IM-MS) [20–23], and oligomer specific antibodies [24–26]. A major analytical challenge is developing a technique which is capable of identification, quantification, and characterization of a wide range of amyloid species.…”