Defining the IsoAspartome

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DESCRIPTION (provided by applicant): Formation of aspartic acid and isoaspartic acid by spontaneous, nonenzymatic degradation of asparagine is THE most common post translational modification in proteins. While deamidation rates have been studied extensively and correlated with many diseases, their breakdown products have not. Isoaspartic acid is generally considered to be worse than aspartic acid since it is generally more destabilizing due to the extra methylene group in the protein backbone chain. Asp/isoAsp formation rates have not been studied due to the difficulty of distinguishing them, but we have recently developed new technology for doing this that will make this research accessible. This project, therefore, has the basic aim of applying our new technology to determining rates of formation of Asp and isoAsp in proteins first on a synthetic peptide level, then on a clean protein level, and finally on a whole-cell proteomics level. This technology involves using Electron Capture Dissociation (ECD) on FTICR mass spectrometers to detect diagnostic marker peaks, c+57, Z-57, and M-60 (see preliminary data) and plot them as a function of time, primary and secondary sequence structure, pH, and temperature thus studying their kinetics and thermodynamics of formation in relevant reaction phase-space. We have already shown that this can be done with individual peptides and proteins, and we expect it to be possible on a proteomics scale using LC/MS/MS experiments where ECD is used as the fragmentation mechanism. The resulting experimental data will be used to update and calibrate theoretical models of deamidation rates to include rates of formation of Asp and isoAsp. A secondary aim is to study the structural dependence of the PIMT repair enzyme (which converts isoAsp to Asp). This enzyme has been used extensively to detect the presence of isoAsp in a wide variety of systems from in vitro peptides to whole tumor cells to studies of erythrocyte aging. Finally, this study will apply these methods to detailed study of isoaspartic acid formation rates via both deamidation and aspartic acid isomerization in bacillus anthracis vaccine protective antigen, monoclonal antibodies used as biopharmaceuticals, and in proteins of interest in cardiovascular disease.