There is a considerable amount of evidence that oxidation of proteins by reactive oxygen species occurs during aging. One of the major objectives of this Program Project is to correlate chemical changes shown to occur in vitro with isolated proteins with oxidative modifications found in aged tissue. Toward this end, the role of several different ROS in the oxidation of CaM, SR and plasma Ca-ATPase, Na+/Ca2+ exchanger and the glutamate binding proteins will be investigated. Although these five proteins differ considerably in their physical properties and biological function, the basic analytical needs to detect the oxidative modifications are the same. Therefore, it is proposed that a Core Facility for the microanalysis of peptides and proteins be set up within the Center for Bioanalytical Research (CBAR) at the University of Kansas. The faculty members of CBAR already have considerable experience in the trace determination of peptides and amino acids, and the existence of this facility within CBAR will provide project members with access to this expertise. The rationale for the Core Facility is based on the following: (1) cost savings by making the instrumentation accessible to all investigators, eliminating redundancies in equipment in individual investigator's laboratories, (2) scientists employed by the Core Facility will spend their time exclusively on the development of analytical methodology for the Program Project, allowing the Program Project members to concentrate on the biological aspects of aging, (3) access to additional instrumentation and expertise within the Center for Bioanalytical Research (4) because of the centralized nature of the facility, it will be easier to incorporate new developments in microanalytical methodology into the Program Project. Because of the potentially small amounts of protein involved in this project, a major emphasis of the facility will be the use of microcolumn-based separation techniques. These include microbore and capillary LC and capillary electrophoresis. Whenever possible, mass spectrometry will be employed as the method of structural identification. However, fluorescence and/or electrochemical detection methods will be employed for identification of specific modifications when appropriate.
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