500nm

1. Chip Chromatography MS2 SDS-PAGE in a chip 6 peptides in 45 sec MS1 RP2 Capillary Array Chromatography IEX 30 sec separation of 6 proteins Multi-dimensional LC-MS/MS RP1 Novel Miniaturized Separations for Proteomics 500nm 200nm Functional Genomic Core The Virtual Institute of Microbial Stress and Survival Rapid Deduction of Stress Response Pathways in Metal/Radionuclide Reducing Bacteria Matthew Fields1,Jay Keasling2, Martin Keller3, Vincent Martin2, Anup Singh4, Dorothea Thompson1, Judy Wall5, Jizhong Zhou1 1Oak Ridge National Laboratory, 2Lawrence Berkeley National Laboratory & University of California, Berkeley, 3Diversa, Inc., 4Sandia National Laboratory, 5University of Missouri, Columbia Overview Protein Complex Identification Microarray hybridization Tandem affinity purification Transcription Profiling • The goal of the FGC is to develop the experimental methods to elucidate the regulatory networks in the stress responses of Desulfovibrio vulgaris, Shewanella oneidensis, and Geobacter metallireducens. In order to accomplish these goals we will: • Use existing DNA arrays for S. oneidensis, and to develop DNA arrays for D. vulgaris and G. metallireducens to measure the transcript profile (transcriptome) during the response to various environmental stresses • Use HPLC-MS-MS to measure the protein profile (proteome) of D. vulgaris, G. metallireducens, and S. oneidensis during the response to various environmental stresses • Measure the metabolite profile (metabolome) of D. vulgaris, G. metallireducens, and S. oneidensis during the response to various environmental stresses • Determine protein-protein interactions in the signaling cascade of D. vulgaris, G. metallireducens, and S. oneidensis during the response to various environmental stresses • Generate mutants in the various genes whose gene products are found to be responsible for a particular stress response, and to compare transcript, protein, and metabolite profiles in the mutant and isogenic wild-type strains and • Biopan the environment for stress response pathways. • The stresses to be examined initially are oxygen, heavy metal exposure (U, Co, Hg, Ni, and Cr), reductant limitation, phosphate restriction, and pH. Label with Cy3 or Cy5 Cultivate, apply stress Profiling of Low-pH Stress Tolerance in S. oneidensis MR-1 Trizol reagent and RNA isolation Transform and select positive clones Incubate 30 min. • Genes Induced at pH 4: ~3,300 genes (~ 60% of total predicted genes) displayed changes in expression in response to 30-minute exposure to pH 4. • outer membrane proteins/lipoproteins • ABC transporter proteins (e.g., phosphate transport system) • metabolic proteins, including those related to amino acid synthesis • hypothetical proteins pH 4 (MES) pH 7 (HEPES) pH 10 (CAPS) Attach “tags” to genes of interest Harvest and suspend cells in buffer Select a pathway involved in stress response Identify protein(s) involved in the pathway to be used as a bait Grown aerobically to mid exponential phase Information on proteins and protein interaction Protein Expression Profiling Graph Database System Quench Metabolism Sonication Ultracentrifugation Stable Intermediates Metabolite Profiling Supernatant Membrane pellet 4M urea Centrifugation Secreted Metabolites HPLC-MS Cells Supernatant Ultracentrifugation Outline of physiological monitoring Lysis Membrane pellet Supernatant Insoluble Fraction GC-MS HPLC-MS Cytoplasm Total Macromolecule Content (DNA, RNA, Lipid, Storage Polymers 1% SDS Urea Soluble Proteins SDS Soluble Proteins Soluble Proteins HPLC-MS CE-MS Derivitization GC-MS Protease Digestions Amino Acid Labeling Pattern Metabolite Amounts And Labeling Patterns 3D LC-MS/MS Review of the Protein Identification from a LCn-MS/MS Run