We propose to characterize 1000 protein forms present in the yeast, Saccharomyces cerevisiae, with unprecedented molecular detail to detect and characterize unexpected mass discrepancies (e.g., covalent modifications) to proteins. This research will combine a completely sequenced genome with recent advances in Fourier-Transform Mass Spectrometry (FTMS) and Electrospray Ionization to translate raw genomic data into biological knowledge and insight. To date, the direct fragmentation of intact proteins has been demonstrated mainly for protein standards or recombinant material. Existing limitations to analyzing intact proteins efficiently include difficulties with solubilization/fractionation, a size constraint, and a lack of dedicated software. However, we will construct a unique measurement platform using an acid-labile surfactant, nanospray robotics, a quadrupole-FTMS Hybrid Instrument, and custom software - all dedicated to realizing measurement throughput. While focusing heavily on technique and software development, it is the biomedical importance of post-translational modifications (PTMs) that drives this research. Targeted for analysis are the 78 proteins from purified yeast ribosomes and 1000 protein forms fractionated directly from its proteome under aerobic and anerobic conditions. In the yeast proteome database, there exist a total of 893 hypothetical and experimentally verified PTMs. We intend to add to this list and enumerate the microheterogeneity, percent occupancy, and dynamic responses of PTMs detected in this study. The research proposed has a pragmatic aspect (i.e., the characterization of unpredictable gene processing events at high detail), but also seeks to establish the analytical infrastructure to eventually contribute in a unique fashion to the Human Proteome Project. The robust detection of eukaryotic PTMs (and quantitation of their changes) is critical to realize the potential of genomic sciences for detecting and fundamentally understanding a broad spectrum of human disease.
Showing the most recent 10 out of 103 publications
