(239c) Separating Early Stage Amyloid Aggregates Using Microchannel Electrophoresis

Authors: 
Redmon, X., University of Arkansas
Hestekin, C. N., University of Arkansas
Moss, M., University of South Carolina
Vance, S. Z., University of South Carolina
Amyloid proteins are involved in a variety of diseases including Alzheimer’s Disease (amyloid beta protein) and Type 2 Diabetes (amylin protein). Several technical challenges exist for the quantitative analysis of different sizes of amyloid proteins present during the early stages of aggregation and thus new technologies are required for enhanced protein separation. Electrophoretic and spectroscopic techniques including SDS-PAGE, western blotting, matrix-assisted laser-desorption ionization mass spectrometry (MALDI-MS), and electrospray ionization (ESI-MS) have been tested for their ability to detect and separate oligomeric Aβ, but have shown limited effectiveness due to oligomeric transience, low concentrations, and difficulty of isolation. In addition, these and other analysis techniques often require the use of excessively high concentrations, denaturing agents, or other components that can alter the native aggregation species. Microchannel electrophoretic offers a fast and efficient separation technique for measuring early Aβ aggregates with little interference to the native aggregation process. A protein’s characteristics, including its tendency to aggregate, are determined largely by its primary sequence, and thus variations in an amyloid’s primary structure can be correlated to changes in its aggregation process. Here we show the results of microchannel electrophoretic analysis of native amyloid beta compared to several primary sequence variants. During native amyloid beta aggregation, monomers and multiple oligomeric species were observed. Comparing variants to native amyloid beta showed that changes in protein flexibility affect protein aggregation rates and propensity to form specific oligomeric species.