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 environmental agents. 2. Determine sites of phosphorylation and other posttranslational modifications on histones that are modulated in cells in response to treatment with environmental agents. 3. 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 domain organization and protein:protein complexes, specifically, the organization of the domains in normal Raf1 and in a constituitively active form and the MLH1:PMS1 protein complex involved in mismatch repair.