This proposal seeks funding for the purchase of a third generation AB/Sciex QTRAP 5500 mass spectrometer. This shared instrument will be used for reliable quantitation of specific posttranslationally modified peptides down to the attomole level - a task for which the QTRAP 5500 was explicitly designed and is uniquely suited. The overall major focus is on studies of the site-specific dynamics and modulation of protein function in response to a variety of cellular cues for this diverse group of biomedical investigations. Sets of covalently modified peptides will be selected for each major project for quantitative measurements by multiple reaction monitoring (MRM). Covalent addition of regulatory and docking moieties (phosphorylation, O-GlcNAcylation, and methylation (mono-, di- and tri-), acetylation and ubiquitination of lysyl e-amino functions, etc.) are enzymatically reversible, but the action of proteolytic enzymes produce irreversible cleavages of particular sets of proteins themselves. The instrument will be housed in the NIH NCRR-supported Mass spectrometry Resource Center at the new Mission Bay Campus at UCSF. This environment assures the availability and involvement of the scientific, technical and management expertise required to optimize its performance and research productivity. The primary theme of the major users focuses on studies of the dynamics of posttranslationally regulated proteins that underlies formation and modulation of signaling pathways and networks as well as the recruitment of protein complexes such as those that regulate chromatin biology. Investigators have major needs in measurements of individual site-specific stoichiometries and their temporal modulation by cellular cues. They are a group of six Principal Investigators involved in some twenty-one NIH supported research programs. These research projects are at the forefront of research in human biology and its disease states. Dr. Charles Craik will chair the Advisory Committee responsible for the overall guidance of the shared instrument's use. Mr. David Maltby will oversee optimization of performance, scheduling, training, maintenance and will provide daily supervision of usage. The close association with the UCSF-NCRR resource and the financial support of the major users will assure long-term productive usage.
Chemical modifications of proteins regulate their function. Impaired regulation of these modifications plays a role in such diseases as HIV, cancer, Alzheimer's disease and epilepsy. Our research goal is to reproducibly quantify levels of protein modification to gain an understanding of the molecular causes of the previously mentioned diseases.
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