Analysis of monomeric Aβ (1–40) peptide by capillary electrophoresis

Abstract

A method was developed to characterize and quantify preparations of monomeric beta-amyloid (Abeta) peptide using capillary electrophoresis (CE) with UV absorbance detection. The detection limit for Abeta monomer using this method was 0.5 microM (19 pg). The self-assembly of Abeta to form amyloid fibrils is closely linked to Alzheimer's disease and is the subject of intense investigations. Consistent preparation of Abeta monomer samples at known concentrations and free of aggregates is a significant challenge for researchers studying the mechanism of Abeta fibril formation and searching for small molecules that inhibit Abeta fibril formation. Samples of Abeta monomer are known to sometimes contain pre-existing aggregates that can affect the kinetics and structure of amyloid fibrils. The CE method presented here showed that some of the monomeric Abeta samples prepared for this study contained a species producing a second peak (in addition to the major monomer peak). The aggregation was monitored using a thioflavin T fluorescence assay, and the resulting fibrils were characterized by transmission electron microscopy. Monomer samples containing the additional peak based on CE analysis were shown to aggregate more rapidly than monomer samples that were free of this putative Abeta aggregate peak.

Gelsolin bound β-amyloid peptides (1–40/1–42) : Electrochemical evaluation of levels of soluble peptide associated with Alzheimer's disease

“…However, most of these methods belong to postmortem identifications, which are often inevitably influenced by the age of the cohort sampled and the method of defining disease pathology (Bennett et al, 2006;Thal et al, 2006). Currently, biochemical assays such as polyacrylamide gel electrophoresis (PAGE), immunoprecipitation, enzyme-linked immunosorbent assay (ELISA) and also non-biochemical techniques, incluindg surface plasmon resonance (SPR), mass spectrometry, capillary electrophoresis have been employed to detect Aβ species from body fluids and cell media (Xia et al, 2010;Golde et al, 2000;Munishkina and Fink, 2007;Picou et al, 2010). Although high sensitivity, selectivity and reliability of ELISA seems more attractive, inherent shortcomings of this technique such as relatively expensive enzyme-linked antibody for Aβ recognition and carcinogenic substrate for chemiluminescent detection should not be ignored.…”

β-Amyloid Biomarker Detection for Alzheimer’s Disease

“…With this electrode they observed a limit of detection of 18 nM (78 ng/mL) and a limit of quantification of 180 nM. The LOD (limit of detection) achieved in this study is better as compared to other ones using other techniques [13]; nevertheless, the LOQ (limit of quantification) was not as good as other studies. Since the levels of Ab in CSF (8-11.9 ng/mL) and plasma (213-290 pg/mL) are lower than the LOD, this technique cannot be applied using body fluids.…”

“…Following rinsing with Tris/HCl buffer, they were then incubated in CuCl2, for copper ions to bind to the Ab deposited in the electrodes. After a second rinse with the buffer, the electrochemical measurements were carried out using a three-electrode system in an electrochemical cell [13]. To compare the measurements of Ab, amylin or islet amyloid polypeptide was used as a control.…”

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].…”