Application Of Mass Spectrometry To Structural Biology

Environmental Health Sciences

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Abstract

Summary of Work:Mass spectrometry is playing a significant role in the identification of unknown proteins at trace levels, in the identification of posttranslational modifications of proteins, and in the identification of proteins that are part of functional/disease related biological complexes. This information can be vital to understanding signal transduction pathways and the regulation of function of the involved proteins. Critical parts of this project are to develop procedures for handling and isolation of sub-picomole levels of proteins, specifically modified proteins (e.g., phosphorylated), intact protein complexes, and their digests in a manner compatible with the final instrumental determinations. Specific aims: 1. Identify the phosphorylation state of histones that are modulated in cells in response to treatment with dexamethasone. 2. Determine sites and levels of phosphorylation and other posttranslational modifications on normal p53 and on p53 phosphorylated in response to specific treatments, such as with environmental stresses and/or by phosphatase inhibitors, and correlate these changes with changes in biological function. 3. Characterize the extracellular domain of the Alzheimer-amyloid precursor protein, including potential intramolecular disulfide bridges, potential phosphorylation and glycosylation. 4. Develop improved techniques for the isolation and detection of phosphorylated proteins. We are currently focusing on improvements in the application of immobilized metal ion affinity techniques and chemical modifications. 5. Develop and apply cross-linking techniques to the characterization of protein:protein complexes.