Pulsed hydrogen–deuterium exchange mass spectrometry probes conformational changes in amyloid beta (Aβ) peptide aggregation

Abstract

Probing the conformational changes of amyloid beta (Aβ) peptide aggregation is challenging owing to the vast heterogeneity of the resulting soluble aggregates. To investigate the formation of these aggregates in solution, we designed an MS-based biophysical approach and applied it to the formation of soluble aggregates of the Aβ 42 peptide, the proposed causative agent in Alzheimer's disease. The approach incorporates pulsed hydrogen-deuterium exchange coupled with MS analysis. The combined approach provides evidence for a self-catalyzed aggregation with a lag phase, as observed previously by fluorescence methods. Unlike those approaches, pulsed hydrogen-deuterium exchange does not require modified Aβ 42 (e.g., labeling with a fluorophore). Furthermore, the approach reveals that the center region of Aβ 42 is first to aggregate, followed by the C and N termini. We also found that the lag phase in the aggregation of soluble species is affected by temperature and Cu 2+ ions. This MS approach has sufficient structural resolution to allow interrogation of Aβ aggregation in physiologically relevant environments. This platform should be generally useful for investigating the aggregation of other amyloid-forming proteins and neurotoxic soluble peptide aggregates.soluble Aβ oligomers | amyloid beta peptide | copper | electrospray ionization | Finke-Watsky mode P rotein aggregation is one of the immediate causes of Alzheimer's, Parkinson, and Huntington diseases, motivating biophysical studies of the responsible proteins. More than 20 small proteins undergo amyloidosis in humans. In Alzheimer's disease (AD), the aggregation of the 40-or 42-aa-long amyloid beta (Aβ) peptide, generally called Aβ 40 or Aβ 42 , respectively, is proposed to be involved in the onset of the disease (1, 2). Aβ 42 is more amyloidogenic and more neurotoxic than Aβ 40 . Although the amyloid-cascade hypothesis suggests that the Aβ-containing amyloid plaques are responsible for neurodegeneration (3-7), other studies suggest that soluble aggregates of Aβ 42 are more neurotoxic than the amyloid plaques (8-13).The amyloid plaques in AD-affected brains contain high levels of copper, zinc, and iron (14-20). Among these, Cu has drawn the most attention because the Aβ precursor protein is likely a Cuchaperone protein (21). Several studies of Cu 2+ -Aβ 40 interactions show that Cu 2+ can promote Aβ 40 aggregation (14,18,19).The structure of Aβ 42 and its aggregates, although studied extensively, remains of high interest. Studies of amyloid fibrils invoke X-ray crystallography (22-24), EM (19,25,26), and thioflavin T fluorescence (19, 27), revealing the polypeptide's global behavior, whereas NMR studies provide residue-level information for the fibrils (28-30). Nevertheless, we know little about soluble Aβ aggregates owing to their intrinsically high heterogeneity.MS should offer an opportunity for investigating soluble aggregates of Aβ 42 . Thus far, there are no MS-based, timedependent studies of the formation of soluble aggregates. Moreover, there are no ot...

Continuous and Pulsed Hydrogen–Deuterium Exchange and Mass Spectrometry Characterize CsgE Oligomerization

“…Continuous HDX is not applicable here, however, as both oligomerization and HDX are time-dependent processes, and the results are a convolution of both. Thus, we applied pulsed HDX, a platform implemented by us previously for Aβ 42 23 , to study CsgE conformational changes accompanying extensive oligomerization.…”

“…Little progress has been made, however, towards understanding CsgE oligomerization. Zhang et al 23 has demonstrated that pulsed HDX is a useful tool for studying soluble Aβ as an aggregation system. Here, we applied this method to follow CsgE conformational changes accompanying oligomerization.…”

Probing the dynamic regulation of peripheral membrane proteins using hydrogen deuterium exchange–MS (HDX–MS)

“…Continuous labelling experiments provide information on the dynamics of the protein studied. A variety of other approaches can also be taken, including pulsed HDX experiments, which primarily are used to measure shortlived folding intermediates [35,37,45,46]. The most common application of HDX-MS has been in the determination of differential exchange rates of proteins upon varying stimuli ( Figure 1B).…”

Structural Transitions and Interactions in the Early Stages of Human Glucagon Amyloid Fibrillation

“…Pulsed HDX with MS analysis has been used successfully to capture the molecular interactions and transient intermediates during fibrillation of other proteins (35)(36)(37)(38). HDX-MS has also been used to characterize glucagon fibrillation at neutral pH (34), though local structural changes during the early stages of fibril formation were not investigated.…”