Simultaneous monitoring of peptide aggregate distributions, structure, and kinetics using amide hydrogen exchange: Application to Aβ(1‐40) fibrillogenesis

Abstract

Increasing evidence indicates that soluble aggregates of amyloid beta protein (Aβ) are neurotoxic. However, difficulty in isolating these unstable, dynamic species impedes studies of Aβ and other aggregating peptides and proteins. In this study, hydrogen-deuterium exchange (HX) detected by mass spectrometry (MS) was used to measure Aβ(1-40) aggregate distributions without purification or modification that might alter the aggregate structure or distribution. Different peaks in the mass spectra were assigned to monomer, low molecular weight oligomer, intermediate, and fibril based on HX labeling behavior and complementary assays. After 1 h labeling, the intermediates incorporated approximately ten more deuterons relative to fibrils, indicating a more solvent exposed structure of such intermediates. HX-MS also showed that the intermediate species dissociated much more slowly to monomer than did the very low molecular weight oligomers that were formed at very early times in Aβ aggregation. Atomic force microscopy (AFM) measurements revealed the intermediates were roughly spherical with relatively homogenous diameters of 30-50 nm. Quantitative analysis of the HX mass spectra showed that the amount of intermediate species was correlated with Aβ toxicity patterns reported in a previous study under the same conditions. This study also demonstrates the potential of the HX-MS approach to characterizing complex, multicomponent oligomer distributions of aggregating peptides and proteins.

The use of mass spectrometry to study amyloid‐β peptides

“…Atomic force microscopy (AFM) has also been applied in combination with HDX/MS to study the various aggregated forms of Ab1-40 and provided details that were undetectable by MS such as size and shape of the aggregates (Qi et al, 2008). Analogously, although MALDI-TOF and ESI/MS could be very useful to characterize a newly synthesized decapeptide with an amino acid sequence contained in the full-length Ab, CD, as well as AFM investigations, were necessary to study the influence of the environmental conditions (the presence of metal ions or sodium dodecyl sulfate (SDS)) on the secondary structure of the peptide to confirm a morphological change before and after copper treatment and/or SDS in the surface packing of the peptide shell (Murariu, Dragan, & Drochioiu, 2007).…”

“…Another experimental method often applied to analyze the different aggregation forms of Ab is represented by combining hydrogen-deuterium exchange (HDX) (reviewed in Tsutsui & Wintrode, 2007) and MS Qi et al, 2008), and MALDI and ESI are both suitable ionization techniques to be applied in this kind of coupled experiments (Kraus, Bienert, & Krause, 2003). The protons that are either involved in H-bonded secondary structures or are buried in a protein's core structure exchange more slowly with deuterium than do solvent-exposed and non-H-bonded protons; therefore, HDX is normally used to investigate secondary structures like amyloid, and some amyloid assembly intermediates that possess a highly stable b-sheet (Kheterpal et al, 2000;Kheterpal, Wetzel, & Cook, 2003).…”

Quantifying Prefibrillar Amyloids in vitro by Using a “Thioflavin‐Like” Spectroscopic Method

“…The ThT signal and kinetics of its appearance correlate well with previous reports under identical conditions. [47] Recombinant ataxin-3 (Q80) was expressed and purified as previously reported. [48] A 75 μM stock solution was diluted to 10 μM in PBS and incubated at 37 °C with shaking for aggregation.…”

Structures of multidomain proteins adsorbed on hydrophobic interaction chromatography surfaces

“…Hydrogen deuterium exchange rates have been shown to be reduced by intermolecular interactions formed in bovine insulin dimers [44], diphtheria toxin oligomers [45], and from aggregation of amyloid beta peptides [46].…”