Capillary electrophoresis for the analysis of the effect of sample preparation on early stages of Aβ_1–40 aggregation

Abstract

Aggregation of the amyloid-β protein (Aβ) contributes to the neurodegeneration characteristic of Alzheimer’s disease. Of particular importance are the early stages of aggregation, which involve the formation of soluble oligomers and protofibrils. In these studies, we demonstrate the potential for CE with UV detection using a polyethylene oxide separation matrix to identify the evolution of various oligomeric species of Aβ1–40. To demonstrate the efficacy of this technique, UV-CE was utilized to compare two methods commonly used to prepare Aβ for aggregation experiments and their effect on the formation of early aggregates. SEC-purified Aβ1–40 initially contained more small species, including monomer, than did freshly dissolved Aβ1–40 pretreated with hexafluoroisopropanol. Strikingly, the lag time to oligomer formation for SEC-isolated Aβ1–40 samples was ~23 h shorter compared to freshly dissolved Aβ1–40 samples. Furthermore, oligomers formed from the aggregation of SEC-purified Aβ1–40 persisted within solution for a longer period of time. These results indicate that the initial sample preparation has a drastic influence on the early stages of Aβ1–40 aggregation. This is the first report of the use of UV-CE with a separation matrix to study the effect of sample preparation on early aggregation of Aβ1–40. UV-CE was also used in parallel with dot blot analysis and inhibitory compounds to discern structural characteristics of individual oligomer peaks, demonstrating the capacity of UV-CE as a complimentary technique to further understand the aggregation process.

Field amplified sample stacking of amyloid beta (1-42) oligomers using capillary electrophoresis

“…A previous study of the aggregation of HFIP treated Ab 1-40 using UV-CE by Pryor et al reported an initial increase in the smaller aggregates before a decrease was observed. 34 A similar pattern can be observed in Fig. 3(a) where the <7 mers species remain high or even increase in concentration, followed by a significant decrease at around 12-18 h of aggregation.…”

“…A 1 mg lyophilized powder of Ab 1-42 sample from AnaSpec (San Jose, CA, USA) was pretreated using 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) as described by Pryor et al 34 The pretreated sample was separated into vials, dried, and stored at À80…”

“…Congo red and Orange G experiments were conducted using 16.7 lM stock solution of inhibitor where 2% of the total sample volume was the inhibitor. This concentration was based on the findings by Necula et al and Pryor et al 34,41 Congo red and Orange G were dissolved in deionized water to make the stock solution.…”

“…33 Furthermore, Pryor et al used UV-CE to demonstrate the differences in Ab 1-40 oligomer aggregation depending on how the samples are prepared. 34 Although electrophoresis in microfluidic chips has been used to study the amyloid beta protein, thus far it has not been used to study its aggregation. 35 Additionally, while these studies have indicated the value and potential for using capillary or microchip electrophoresis to improve our understanding of FIG.…”

Advances and Pitfalls in the Capillary Electrophoresis Analysis of Aggregates of Beta Amyloid Peptides

“…CE with UV detection was employed for the analysis of several Aβ monomers [16,17], for the separation of monomers and transient soluble oligomers of Aβ 1-40 or 1-42 using neutral saline background electrolyte [18][19][20][21] or a polymeric separation matrix [22]. Moreover, CE-UV allowed the separation of monomers, large aggregates (migrating as wide peak) and fibrils (migrating as sharp peaks or spikes) [23].…”