Desulfovibrio vulgaris Hildenborough (DvH) is a sulfate

1. Identification and mapping of post translational modifications involved in stress response in Desulfovibrio vulgaris Hildenborough SOOH Thioredoxin peroxidase (cytoplasmic) SOH SH S S DsbA (periplasmic) H2O2 Free Thiol H-S-Glutathione(cytoplasmic) S-Glutathione Proteomics Approach to Understanding Stress Response Introduction ICAT for PTM Survey of Redox Active Thiols Post-translational Modifications (PTM) in Stress Response Proteomics to Identify Proteins Phosphorylated During Stress Acknowledgements Proteomics Techniques for PTM Survey and Analyses S V V V V V C C C C C C1 control baseline T0 V1 stress Growth (OD) 0.3 0 2 Time (hours) Rajat Sapra1,2 , Sara Gaucher1,2, Gabriela Chirica1, George Buffleben1, Arlene Gonzales1, Carrie Muesberger1, and Anup Singh1,2 1Biosystems Research Department, Sandia National Laboratories, Livermore, CA 94551, 2Virtual Institute of Stress and Survival. IAM = Light Chain IAM = Heavy Chain Desulfovibrio vulgaris Hildenborough (DvH) is a sulfate reducing bacterium that grows in the absence of oxygen.   From a physiological, as well as ecological, standpoint anaerobic bacteria have to overcome oxygen stress.   To counter the effects of oxygen stress many of the anaerobic bacteria have evolved different mechanisms for 'oxygen detoxification'. We are investigating the proteomic response of DvH in response various stresses including Oxygen stress and the resulting proteome regulation and post translational modification of proteins using Difference In-Gel Electrophoresis (DIGE), in conjunction with Isotope Coded Affinity Tag (ICAT). The kinetics of proteomics response to oxygen stress and the post translational modification of proteins, specifically glycosylation and phosphorylation is being investigated using a combination of DIGE and MALDI to identify the proteins and LC-MS to map the sites of modification. ICAT is being used for the investigation of the sulfhydryl groups in Cys residues that may be modified in oxygen and oxidative stress. Common Post Translational Modifications of Thiol Containing Proteins Comparative Proteomics: Differential In-Gel Electrophoresis is used To compare the different proteomic samples to identify the proteins that are regulated in stress response. Standard-Label with Cy2 Sample 1- Label with Cy3 Sample 3- Label with Cy5 Pool Samples 2-D electrophoresis Scan 1 Scan 2 Scan 3 Overlay Current Model for Oxygen Detoxification in Desulfovibrio species Using ICAT To Track Thiol Disulfide States In Proteins: The disulfide bonds in the control and the variable samples are differentially labeled with ICAT light and heavy labels. The labeled and processed proteins results in labeled peptides which are analyzed to identify proteins where a free thiol has been modified to disulfide linkage. The advantages of ICAT are that the data generated it is complimentary to DIGE, we can get quantitative information about the state of the thiol modifications and overall it may help track lower abundance proteisn that may be modified in response to the stress. pH4 pH7 Time 1 = 30min # of Proteins Upregulated = 15 # of Proteins Downregulated = 30 # of Proteins Upregulated = 24 Time 2 = 60 min 4. Rubredoxin 5. Cytochrome containing enzymes 6. Glutaredoxin; 7. NADH peroxidase • Redox Enzymes • Superoxide dismutase • Rubredoxin oxidoreductase # of Proteins Downregulated = 68 Time 3 = 120 min # of Proteins Upregulated = 32 Isolate Control Cell Samples Lyse Cells Compare Proteome # of Proteins Downregulated = 30 Isolate Stress Cell Samples Lyse Cells Cell Lysate Time 4 = 240 min # of Proteins Upregulated = 33 Column Purification of Phosphorylated Proteins 2D-Electrophoresis Of The Lysate 1 2 3 4 5 6 7 8 Phospho-Protein Specific Stain # of Proteins Downregulated = 30 Total Protein Stain Modification Type Common Function Modification The DvH proteome undergoes regulation at the at the surveyed time points in response to oxygen. The proteins regulation is maximum at the 60 minute time point where a total of 88 proteins spots are regulated more than 2 fold, which decreases to 62 and 63 protein spots at the 120min and 240 min time points, respectively. The initial response of the proteome is seen to be slow as a total of 45 protein spots are regulated and it is hypothesized that the initial response may be related to post translational modifications of proteins to counter the oxygen stress. Phophorylations Phosphoryl group Nutrient stress Comparative Analysis and Identification Structural stress, temperature stress Glycosylation Sugar group Thiol-disulfide modifications Redox Interconversion of thiols and disulfide Redox stress, Structural stress This work was part of the Virtual Institute for Microbial Stress and Survival supported by the U. S. Department of Energy, Office of Science, Office of Biological and Environmental Research, Genomics Program: GTL through contract DE-AC03-76SF00099 between Lawrence Berkeley National Laboratory and the U. S. Department of Energy.