A data treatment method for detecting fluorescence anisotropy peaks in capillary electropherograms.

A data treatment method is presented to detect fluorescence anisotropy (FA) peaks in capillary electrophoresis electropherograms. The data treatment method converts plots of fluorescence anisotropy vs. time that contain no peaks that are distinguishable from the noise of the anisotropy background into plots that show distinct fluorescence anisotropy peaks. The method was demonstrated using laser-induced fluorescence anisotropy data from individual Aß (1-42) aggregates separated using capillary electrophoresis. Applying this data treatment method enabled the detection of anisotropy peaks for individual Aß aggregate fluorescence peaks that were not observed prior to the data treatment method. The data treatment method is not specifically designed for Aß aggregate analysis or capillary electrophoresis, and it should be applicable to other applications and other separation methods with FA detection.

Separation and detection of individual Aß aggregates by capillary electrophoresis with laser-induced fluorescence detection.

The separation and detection of individual amyloid beta (Aß) aggregates by capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) was demonstrated. Samples were prepared with either Aß (1-40) or Aß (1-42) peptides and were characterized by CE with ultraviolet (UV) absorbance detection and transmission electron microscopy (TEM). Using thioflavin T (ThT) in the electrophoresis buffer, electrophoresis of aggregate-containing samples (5.0-s injection) produced up to several hundred narrow (< 20 ms FWHM [full width at half maximum]) fluorescence peaks. Injection of Aß (1-40) monomer samples resulted in no additional peaks compared with controls. The CE-LIF results were validated by bulk ThT fluorescence measurements for the same samples. The potential of laser-induced fluorescence anisotropy (LIFA) with CE to characterize individual Aß aggregates also was investigated.

Analysis of Aß (1-40) and Aß (1-42) monomer and fibrils by capillary electrophoresis.

A method based on capillary electrophoresis (CE) with UV absorbance detection is presented to characterize synthetic amyloid beta (Aß) peptide preparations at different aggregation states. Aggregation of Aß (1-40) and Aß (1-42) is closely linked to Alzheimer's disease (AD), and studying how Aß peptides self-assemble to form aggregates is the focus of intense research. Developing methods capable of identifying, characterizing and quantifying a wide range of Aß species from monomers to fully formed fibrils is critical for AD research and is a major analytical challenge. Monomer and fibril samples of Aß (1-40) and Aß (1-42) were prepared and characterized for this study. The monomer-equivalent concentration for each sample was determined by HPLC-UV, and aggregate formation was confirmed and characterized by transmission electron microscopy. The same samples were studied using CE with UV absorbance detection. Analysis by mass spectrometry of collected CE fractions was used to confirm the presence of Aß for some CE-UV peaks. The CE-UV method reported here clearly indicates that monomeric and aggregated Aß were electrophoretically separated, and substantial differences in the electrophoretic profiles between samples of Aß (1-40) and Aß (1-42) were observed. This CE-UV method can differentiate between Aß monomer, oligomeric intermediates, and mature fibrils.

Analysis of monomeric Abeta (1-40) peptide by capillary electrophoresis.

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.