Application Of Mass Spectrometry To Structural Biology

Tomer, Kenneth

Abstract

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.

Funding Agency

• Agency: National Institute of Health (NIH)
• Institute: National Institute of Environmental Health Sciences (NIEHS)
• Type: Intramural Research (Z01)
• Project #: 1Z01ES050127-10
• Application #: 6672993
• Study Section: (LSB)
• Support Year: 10
• Fiscal Year: 2002

Institution

• Name: U.S. National Inst of Environ Hlth Scis
• Country: United States

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

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

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

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