(597d) The Use of Capillary Electrophoresis to Monitor the Early Stages of Insulin Aggregation
Protein folding abnormalities contribute to many amyloid diseases such as: Alzheimer's disease, Parkinson's disease, Type II diabetes mellitus, prion diseases, and Huntington's disease. Insoluble protein aggregates, termed amyloids, which exhibit a cross beta-sheet structure are a common characteristic of amyloid diseases. The molecular mechanism by which proteins progress from monomer to oligomer to insoluble amyloid aggregates is not well understood. The formation of low molecular weight oligomeric species during the early stages of protein misfolding is of particular interest for disease prevention. Currently, diagnostic techniques which monitor the very early stages of protein misfolding remain inadequate. Insulin protein is known to form amyloid aggregates displaying a cross beta-sheet structure. In this work, capillary electrophoresis is used to monitor the progression of insulin protein from monomeric to low molecular weight oligomeric species. A critical aggregation time period of 8 to 10 hours was found to be necessary for the formation of low molecular weight oligomeric species, however this time period was dependent on aggregation conditions such as the amount of salt. The limit of detection of insulin using capillary electrophoresis detection by both UV and laser induced fluorescence was determined. By determining the early aggregation kinetics of insulin at physiologically relevant concentrations (nM-pM range), we have shown that capillary electrophoresis has the ability to detect other disease causing amyloid proteins, similar to insulin, present in cerebrospinal fluid or plasma.