Ammonium hydroxide treatment of Aβ produces an aggregate free solution suitable for biophysical and cell culture characterization

Abstract

Alzheimer’s disease is the leading cause of dementia in the elderly. Pathologically it is characterized by the presence of amyloid plaques and neuronal loss within the brain tissue of affected individuals. It is now widely hypothesised that fibrillar structures represent an inert structure. Biophysical and toxicity assays attempting to characterize the formation of both the fibrillar and the intermediate oligomeric structures of Aβ typically involves preparing samples which are largely monomeric; the most common method by which this is achieved is to use the fluorinated organic solvent 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP). Recent evidence has suggested that this method is not 100% effective in producing an aggregate free solution. We show, using dynamic light scattering, size exclusion chromatography and small angle X-ray scattering that this is indeed the case, with HFIP pretreated Aβ peptide solutions displaying an increased proportion of oligomeric and aggregated material and an increased propensity to aggregate. Furthermore we show that an alternative technique, involving treatment with strong alkali results in a much more homogenous solution that is largely monomeric. These techniques for solubilising and controlling the oligomeric state of Aβ are valuable starting points for future biophysical and toxicity assays.

A routine method for cloning, expressing and purifying Aβ(1–42) for structural NMR studies

“…At the high concentrations required, it is strictly necessary that pre-aggregated material is removed; otherwise, seeded fibrillization would lead to rapid loss in signal. Ammonia pretreatment, followed by dissolution into sodium hydroxide (Ryan et al 2013) worked effectively for this purpose. Figure 4 displays a well-resolved HSQC spectrum of monomeric Aβ(1-42).…”

ThT 101: a primer on the use of thioflavin T to investigate amyloid formation

“…Cleavage and purification of synthetic peptides typically involves trifluoroacetic acid (TFA), leading to TFA salts of the peptide. These are often present in lyophilized peptide preparations, along with chemical scavengers (chemical compounds added to the reaction in order to remove or de-activate impurities and unwanted reaction products) and can complicate the solubility and preparation of peptide stock solutions in physiological buffers [57][58][59].…”

“…For Ab peptides, a widely used method to dissolve/prepare peptides for fibril formation experiments involves pretreating synthetic peptides with NaOH (or alkaline solutions) [51,58,[68][69][70]. This method has also been used for a-synuclein [71] and is recommended by vendors for acidic peptides [72].…”

“…However, the transition from acidic (pH %2) to neutral physiological buffers usually passes through the isoelectric point of the peptide where the aggregation propensity is maximal and solubility is minimal. This is avoided by alkaline dissolution, resulting in higher yields of monomeric Ab peptide and low content of aggregates [51,58,68,69]. Another option is the use of aqueous solution of ammonium hydroxide, which is volatile and therefore easy to lyophilize away.…”

Quantitative detection of amyloid-β peptides by mass spectrometry: state of the art and clinical applications

“…During their re-suspension, Aβ peptides might however go back to the beta sheet conformation they had prior to freeze-drying and thus could rapidly form new aggregates [70]. To overcome this hurdle, standards are often pretreated with hexafluoroisopropanol (HFIP) [71] in order to denature beta sheets and favor α-helix conformations. Aβ standards need eventually to be re-suspended in solvents compatible with MS analysis, i.e., acetonitrile/ammonium hydroxide (20%:1%, v/v) [42,66] or dimethylsulfoxide (DMSO) [40].…”