One of the primary consequences of the exposure of proteins to reactive oxygen species (ROS) is the covalent modification of proteins. Two ionization methods have revolutionized protein mass spectrometry; electrospray (ESI) and matrix-assisted laser desorption (MALDI). The Facility for Microanalysis of Peptides and Proteins has successfully applied capillary HPLC and capillary electrophoresis to the analysis of program project proteins coupled with both on and off-line detection by MALDI, ESI and FAB MS. The Core Facility has been utilized for the identification of oxidation and nitration sites in the target proteins calmodulin (CaM), CaATPase, and a peptide corresponding to the active site of the glutamate binding protein. It was found that CaM oxidizes exclusively at methionine. Analysis of CaM showed and age-correlated increased in methionine oxidation, but the oxidation is not complete. The oxidized CaM can be repaired in vitro with methionine sulfoxide reductase resulting in the recovery of function. Nitration sites were identified in CaATPase isolated from aged rat, and the in vitro oxidative susceptibility of cysteine sites on CaATPase in a native state was investigated. The disulfide status of cysteines in SERCA were determined with a thiol alkylation strategy that will be incorporated into the Core. It was found that a ubiquitous marker of protein oxidation may be methylene lysine. Several new instruments are available to Core B. These include a triple quadrupole (QQQ), a tandem quadrupole time-of-flight OF) and a high resolution MALDI-TOF. The QQQ will be used for quantitation of amino acids. The Q-TOF will be used for the high sensitivity mapping of ROS modified peptides. The MALDI-TOF will be used to apply mass spectrometric based proteomic strategies for high sensitivity target protein identification during isolation of trace amounts of protein. The high resolution and high sensitivity capability of the Q- TOF will be used to develop a rapid and sensitive method for measuring the oxidation state of small proteins isolated from aged tissue and cells. Installation of the MALDI-TOF and QQQ are complete, the Q-TOF will be installed August 2000. Preliminary data on PMCA was obtained on the MALDI-TOF and data on project related samples was acquired during demonstrations of the QQQ and Q-TOF.
Showing the most recent 10 out of 171 publications
