Abstract

of WorkMass 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.Correlate surface accessibility with molecular modeling to provide tertiary structure prediction.2.Determine sites of interaction between proteins and DNA based on cross-linking an MS/MS identification of cross-linking sites and on protection assays.3.Characterize the regulatory domain of PP5 and its site of interaction.4.Elucidation of phosphorylation sites on p53 and of the interrelationship between phosphorylation of specific sites and biological activity. Biological processes often involve specific non-covalent and/or covalent interactions. Understanding the structures of functional intermediates and products is important in determining biochemical mechanisms and how specific molecules interact with endogenous compounds.
Specific aims :1.Identify the amino acids of b-polymerase that can form the covalent intermediate with the abasic site in excision-repair.2.Identify amino acids phosphorylated on histones that are modulated in cells in response to treatment with dexamethasone.3. 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.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of Environmental Health Sciences (NIEHS)
Type
Intramural Research (Z01)
Project #
1Z01ES050127-08
Application #
6432348
Study Section
(LSB)
Project Start
Project End
Budget Start
Budget End
Support Year
8
Fiscal Year
2000
Total Cost
Indirect Cost

  Institution

Name
U.S. National Inst of Environ Hlth Scis
Department
Type
DUNS #
City
State
Country
United States
Zip Code

  Publications

Schorzman, Allison N; Perera, Lalith; Cutalo-Patterson, Jenny M et al. (2011) Modeling of the DNA-binding site of yeast Pms1 by mass spectrometry. DNA Repair (Amst) 10:454-65
Dhungana, Suraj; Fessler, Michael B; Tomer, Kenneth B (2009) Epitope mapping by differential chemical modification of antigens. Methods Mol Biol 524:119-34
Perdivara, Irina; Deterding, Leesa; Moise, Adrian et al. (2008) Determination of primary structure and microheterogeneity of a beta-amyloid plaque-specific antibody using high-performance LC-tandem mass spectrometry. Anal Bioanal Chem 391:325-36
Iacob, Roxana E; Keck, Zhenyong; Olson, Oakley et al. (2008) Structural elucidation of critical residues involved in binding of human monoclonal antibodies to hepatitis C virus E2 envelope glycoprotein. Biochim Biophys Acta 1784:530-42
Clark, Alan B; Deterding, Leesa; Tomer, Kenneth B et al. (2007) Multiple functions for the N-terminal region of Msh6. Nucleic Acids Res 35:4114-23
Sharp, Joshua S; Tomer, Kenneth B (2007) Analysis of the oxidative damage-induced conformational changes of apo- and holocalmodulin by dose-dependent protein oxidative surface mapping. Biophys J 92:1682-92
Prasad, Rajendra; Liu, Yuan; Deterding, Leesa J et al. (2007) HMGB1 is a cofactor in mammalian base excision repair. Mol Cell 27:829-41
Venkatesh, Sanjay; Tomer, Kenneth B; Sharp, Joshua S (2007) Rapid identification of oxidation-induced conformational changes by kinetic analysis. Rapid Commun Mass Spectrom 21:3927-36
Robinette, David; Neamati, Nouri; Tomer, Kenneth B et al. (2006) Photoaffinity labeling combined with mass spectrometric approaches as a tool for structural proteomics. Expert Rev Proteomics 3:399-408
Sharp, Joshua S; Sullivan, Daniel M; Cavanagh, John et al. (2006) Measurement of multisite oxidation kinetics reveals an active site conformational change in Spo0F as a result of protein oxidation. Biochemistry 45:6260-6

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