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Bob Hettich PhD: Analytical Chemistry, Purdue University (1986) Oak Ridge National Laboratory |
Research Area:
The primary focus of my research is the high-resolution detection and structural characterization of important biomolecules, such as proteins and protein variants, by mass spectrometry. The principal tools used for this work are Fourier transform ion cyclotron resonance (FTICR) mass spectrometers, equipped with either MALDI or electrospray ionization sources.
Current Students & Research Projects
Brian Erickson: An Integrated "Top-down" and "Bottom-up" Mass Spectrometric Analysis of Protein Strain Variation in a Natural Microbial Proteome.
Carlee McClintock: Topologically Informative Residues of Large Proteins Can Be Marked by Oxidation and Mapped by Mass Spectrometry.
Alison Russell: Analysis of the Human Gut Microbiome.
Melissa R. Thompson: Dosage-Dependent Proteome Response of Shewanella Oneidensis MR-1 to Chromate Insult.
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Description of Research:
The advent of MALDI and electrospray ionization sources for mass spectrometry has revolutionized the application of this technology for biological studies. In particular, large biomolecules (proteins, oligonucleotides, protein:protein complexes) can now often be studied directly with mass spectrometry. This has provided biologists with an additional tool to study many biological systems at the molecular level. At ORNL, we have a substantial research effort in the Biological and Organic Mass Spectrometry Group to develop and evaluate mass spectrometry technology for a variety of biological problems. This research group consists of six staff members, who direct individual research areas with a variety of mass spectrometry instrumentation (quadrupole ion traps, triple quadrupoles, time-of-flight, and Fourier transform ICR).
My research interests focus on sensitive detection and structural characterization of biomolecules with ES-FTICR-MS and MALDI-FTICR-MS. A new, high performance ES-FTICR-MS instrument was installed at ORNL in March 2000. This instrument will be upgraded with a higher field magnet (9.4 T) in September 2000, and will provide unprecedented capabilities for high-resolution measurement and ion interrogation. These FTICR mass spectrometers provide the highest resolution mass measurement currently possible, which is very useful for examining biomolecules at the molecular level. In addition to accurate mass measurement under high-resolution conditions, the ion trapping ability of FTICR-MS provides a means to investigate ion structures. In particular, collisional dissociation and ion-molecule reactions (such as hydrogen/deuterium exchange) can be conducted in the gas phase inside the mass spectrometer, and provide important information about ion structures. For example, it is often possible to use collisional dissociation methods to identify a sequence tag (i.e. short amino acid sequence section) from a protein. The combination of this information, along with the accurate molecular mass, is often sufficient to unambiguously identify the protein from a database, even if the complete sequence has not been determined. The ability of mass spectrometry to measure and identify biomolecules even in mixtures (such as a protein digest) enables rapid compound identification with minimal sample cleanup.
Research in our lab is also moving into the area of examining higher order protein structure with mass spectrometry. For example, gas phase H/D exchange reactions provide information into the secondary structure of proteins, revealing how their gas phase structures are related to solution phase structures. Preliminary work has also indicated that mass spectrometry may be useful for examining protein:protein and protein:DNA interactions. For this issue, complexes such as ligand-receptor systems are generated in the solution phase, and then transferred to the mass spectrometer via the electrospray process. This research is not routine at this point, as many details concerning non-specific binding, gentle ionization conditions, structure alteration, etc. need to be more fully explored. However, the ability of mass spectrometry to examine proteins and oligonucleotides, as well as their complexes, has lead to vigorous research activity not only at ORNL, but also at many other mass spectrometry research laboratories world-wide.
Selected Publications:
- Hettich RL Investigating the Effect of Transition Metal Ion Oxidation State on Oligodeoxyribonucleotide Binding by MALDI-FTICR-MS. Int J Mass Spec. 2001;204:55-75.
- Ji HF, Dabestani R, Brown GM, Hettich RL. Synthesis and sensing behavior of cyanoanthracene modified 1,3-alternate calix[4]benzocrown-6: a new class of Cs+ selective optical sensors. Journal of the Chemical Society-Perkin Transactions. 2001;2:585-91.
- Uchiki T, Hettich RL, Gupta V, Dealwis C. Characterization of Monomeric and Dimeric Forms of Recombinant Sml1p-histag Protein by Electrospray-MS. Anal. Biochem 2002;301:35-48.
- Kertesz V, Deng H, Asano KG, Hettich, RL, Van Berkel GJ. N-Phenyl-1,4-Phenylenediamine and Benzidine Oxidation Products Identified Using On-Line Electrochemistry/Electrospray Fourier Transform Mass Spectrometry. Electroanalysis. 2002;14:1027-30.
- VerBerkmoes NC, Strader MB, Smiley D, Howell EE, Hurst GB, Hettich R L, Stephenson, Jr JL. Intact Protein Analysis for Site Directed Mutagenesis Overexpression Products: Plasmid Encoded R67 Dihydrofolate Reductase. Anal Biochem. 2002;305:68-81.
- VerBerkmoes NC, Bundy JL, Hauser L, Asano KG, Razumovskaya J, Larimer F, Hettich RL, Stephenson, Jr. JL. Integrating "Top-Down" and "Bottom-Up" Mass Spectrometric Approaches for Proteomic Analysis of Shewanella oneidensis. J Proteome Research. 2002;1:239-52.
- Sharp J, Becker J, Hettich RL. Protein Surface Mapping by Chemical Oxidation: Structural Analysis by Mass Spectrometry. Anal Biochem. 2003;313: 216-25. 10.
- VerBerkmoes N, Sharp J, Hettich RL. "Mass Spectrometry" in Microbial Functional Genomics, edited by Jizhong Zhou and Dorothea Thompson, John Wiley & Sons, 2004.
- Keller K, Brodbelt J, Hettich RL, Van Berkel GJ. Comparison of Sustained Off-Resonance Irradiation Collisionally Activated Dissociation and Multipole Storage-Assisted Dissociation for Top-Down Protein Analysis. J Mass Spec. 2004;39:402-11.
- "Identification of Phosphorylation Sites on the Yeast Ribonucleotide Reductase Inhibitor Sml1," J. Biol. Chem. (2004), 279, 11293-11303.
- Ross S, Srinivas P, Clifford A, Lee S, Philbert M, Hettich RL. New Technologies for Nutrition Research. J Nutrition. 2004;134:681-85.
- Gupta V, Peterson C, Dice L, Uchiki T, Racca J, Guo J, Xu Y, Hettich RL, Zhao X, Rothstein R, Dealwis C, Sml1p is a Dimer in Solution: Characterization of Denaturation and Renaturation of Recombinant Sml1p. Biochemistry. In press for summer 2004.
- Razumovskaya J, Olman V, Xu D, Uberbacher E, VerBerkmoes N, Hettich RL, Xu Y. A Computational Method for Assessing Peptide-Identification Reliability in Tandem Mass Spectrometry Analysis with Sequest. Proteomics. 2004;4:961-9.
- Strader M, VerBerkmoes N, Tabb D, Connelly H, Barton J, Bruce B, Pelletier D, Davison B, Hettich RL, Larimer F, Hurst G. Characterization of the 70S Ribosome from Rhodopseudomonas palustris Using an Integrated "Top-Down" and "Bottom-Up" Mass Spectrometric Approach. J Proteome Res. In press for summer 2004.
- Sharp J, Becker J, Hettich RL. Analysis of protein solvent accessible surfaces by photochemical oxidation and mass spectrometry. Anal Chem. 2004;76:672-83.