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Bob Hettich PhD: Analytical Chemistry, Purdue University (1986) Oak Ridge National Laboratory |
Student Project Abstracts:
An Integrated "Top-down" and "Bottom-up" Mass Spectrometric Analysis of Protein Strain Variation in a Natural Microbial Proteome
Brian Erickson; Mark Lefsrud; Nathan VerBerkmoes; Steven Singer; Michael Thelen; Jillian Banfield; Robert Hettich
Recent technological advancements in mass spectrometry have vastly improved the ability to identify and characterize the proteome of a microbe. Unfortunately, much of the current research is centered on culture-based studies which limit the ability to identify critical environmental interactions and strain variants. Historically, intact protein (“top-down”) measurements and protease-digested protein (“bottom-up”) measurements have been two separate and distinct methods. “Top-down” mass spectrometry enables researchers to obtain a clearer picture of intact protein isoforms, which can include post-translational modifications and sequence variants. On the other hand, “bottom-up” mass spectrometry allows for a broader view of the proteome, resulting in a catalog of proteins present in the microbial community. Integration of both techniques will aid in overcoming many of the inherent problems encountered when studying microbial communities, such as easy peptide to protein identification and assignment of proteins to originating species. The integrated “top-down” and “bottom-up” technique was applied to the extracelluar fraction of cells from an acid mine drainage (AMD) microbial biofilm community. Notably, the microbial biofilm community from the AMD exists in pH < 1, at ~42° C, with molar/sub-molar concentrations of metals. Given the extreme nature of the environment and the presence of multiple species present in the biofilm, the extracellular fraction is ideal for detailed analysis of the proteins present and their possible variants. Preliminary “bottom-up” mass spectrometric measurements of three related but distinct HPLC cytochrome fractions revealed a variety of characteristic peptides that provided high sequence coverage for the cytochrome-579 protein. Intact proteins from the same original samples were examined by direct infusion ESI-FTICR-MS. Molecular mass measurements revealed the presence of at least three abundant isoforms of the cytochrome 579 species, at Mr = 14316, 15690, and 16243 Da. Each was subjected to infrared multiphoton dissociation (IRMPD) in the ICR cell and high resolution measurements of the fragment ions revealed two dominant sequence tags for an interior region: MVWVVSNGS as expected based on the leptosprillium II genome data, and an unexpected variant version: MFWVVANGS. This top-down information was used to re-search the bottom-up datasets for specific variants, which confirmed the presence of both versions.
Topologically Informative Residues of Large Proteins Can Be Marked by Oxidation and Mapped by Mass Spectrometry
Carlee McClintock, Christine Shook, Vilmos Kertesz, Gary Van Berkel, Robert L. Hettich
Oxidative footprinting methods have been developed to map the solvent accessible regions of proteins by pinpointing covalently labeled surface residues with tandem mass spectrometry. This approach is particularly useful for obtaining structural information on large proteins that are difficult to crystallize and exceed the size limitation for NMR structural studies. We have applied this technique to physiologically important human (HSA) and bovine (BSA) serum albumin homologs and compared the results to homology models built for BSA based on the resolved HSA crystal structure using MOE software. Buffered solutions were prepared with 0.5 mg/mL protein and 1-10% hydrogen peroxide, then UV-irradiated for 1-5 minutes. The reaction was quenched by catalase and oxidized proteins were denatured and reduced prior to trypsin digestion overnight. Peptides were extracted and analyzed by reverse-phase LC-MS/MS, and candidate peptides scored by the MASPIC algorithm were reported as normal or oxidized by the peptide search algorithm DBDigger. A comparative analysis of shared and unique modifications between the denatured and native samples separates intrinsically reactive residues that do not provide useful solvent accessibility information from those that do. The variation in amino acid composition between BSA and HSA, which share 76% sequence identity, provides an opportunity to compare the reactivity of different residues in similar chemical environments. Approximately 30% of over 580 amino acids in these model proteins are theoretically susceptible to oxidation by hydroxyl radical attack. Of nearly 200 potentially reactive sites, about half are calculated by GETAREA for the HSA atomic coordinates to be suitably exposed for oxidative labeling, whereas reactive residues buried in the native conformation typically remain unmodified, with the exception of methionine. Oxidation of denatured proteins revealed significant but incomplete oxidation levels that serve as controls against false negatives in the experimental datasets. Manually verified search results for these two proteins reveal oxidized sites in overlapping peptides that corroborate solvent accessible regions in the known HSA structure.
Dosage-Dependent Proteome Response of Shewanella Oneidensis MR-1 to Chromate Insult
Melissa R. Thompson, Nathan C. VerBerkmoes, Karuna Chourey, Steven D. Brown, Robert L. Hettich, and Dorothea K. Thompson
Shewanella oneidensis MR-1, a gram-negative, facultatively anaerobic bacterium can utilize metal ions alternative terminal electron acceptors during anaerobic respiration. Our goal is to identify molecular components of chromate resistance and biotransformation response pathways to assess the potential of MR-1 for bioremediation. Previous work with cells exposed acutely and chronically to chromate reveals protein expression changes, with those putatively involved in chromate detoxification and stress response present at a higher level. The goal of this study is to compare the initial proteome response of S. oneidensis cells to sub-lethal doses of chromate. MR-1 cells were grown aerobically in LB to OD600 0.5, shocked with 0.3, 0.5, and 1.0 mM K2CrO4, and harvested 30 min later. In each case, cells were lysed by sonciation followed by separation into 2 protein fractions (soluble and membrane). Protein fractions were trypsin digested and analyzed via multidimensional HPLC-NanoESI-MS/MS utilizing a LCQ Deca XP Plus operated in the data dependent mode using SEQUEST for peptide identification. Up- or down-regulated proteins were identified using semi-quantitation taking into account protein sequence coverage, peptide count, and spectral count. The criteria are: > 30% sequence coverage, > 4 unique peptides, and/or 2X more MS/MS spectra identified. Up-regulated proteins found after acute chromate exposure (45 and 90 min post shock) include a putative azoreductase (SO3585), glyoxylase (SO3586), and a membrane-associated hypothetical protein (SO3587) organized into a predicted operon and metal ion uptake proteins (TonB receptors, AlcA, TonB, and ExbB). 1150 non-redundant proteins were identified from a combination of the control and 3 dosage measurements, with each averaging ~630-860 proteins. AlcA and a putative TonB-dependent receptor are up-regulated in the dosage conditions as well as in the acute study. Interestingly, under the dosage conditions presented here, preliminary results suggest expression of some proteins is unique to the chromate dosage, and may not exactly parallel the results obtained by microarray analysis.