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What is Desulfovibrio vulgaris ?

Modificomics! Redox Proteomics In Desulfovibrio vulgaris Hildenborough: Search For Proteins That Mediate Stress Response via Post-Translational Modification of Cys residues. Rajat Sapra and Sara Gaucher Sandia National Labs. What is Desulfovibrio vulgaris ?.

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What is Desulfovibrio vulgaris ?

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  1. Modificomics! Redox Proteomics In Desulfovibrio vulgaris Hildenborough: Search For Proteins That Mediate Stress Response via Post-Translational Modification of Cys residues.Rajat Sapra and Sara GaucherSandia National Labs

  2. What is Desulfovibrio vulgaris? • Anaerobic sulfate reducing bacteria (SRB) • Metabolism • Model sulfate reducing bacterium • Energy source- respiratory reduction of sulfate • Economic Impact • Biocorrosion of ferrous metals in the environment • DOE organism  GTL • Can reduce soluble U(VI) to insoluble U(IV) • Other toxic metals like Cr (VI) Cr(III) • Sequenced genome • Size: Chromosome 3.57 Mb/ Plasmid 0.20 Mb • Number of genes: Chromosome 3480/ Plasmid 152

  3. e- e- e- e- O2 O2- H2O2 OH. H2O 1 2 3 4 2 e- H2O2 H2O + O2 5 H2O2 + O2-. OH. + OH- + O2- Oxygen and Anaerobes • Problem for anaerobes: • Abundant supply of reductant (electrons)- anaerobes are in a reducing environment • Fenton Reaction (Fe2+ + H2O2  Fe3+ + OH- + OH. ) • Fe is everywhere: inside and outside the cell

  4. Current Model for Oxygen Detoxification in Desulfovibrio species H2O2 1 2 + O2 H2O2 O2-. O2 O2 O2 O2 O2 O2 1 5 O2-. H2O H2O2 H2O 4 3 6, 7? Cytoplasm Membranes Periplasm • Redox Enzymes • Superoxide dismutase • Rubredoxin oxidoreductase 4. Rubredoxin 5. Cytochrome containing enzymes 6. Glutaredoxin; 7. NADH peroxidase

  5. Post-translational Modifications (PTM) in Stress Response • Post-translational modifications are modification(s) in the protein post production • PTMs can be reversible or irreversible • PTMs abundant in nature • Used in signaling, recognition and stress response pathways • (among other functions) Some common PTMs that we would expect in DvH: Modification Modification Type Common Function 1. Phophorylations Phosphoryl group Nutrient stress Structural stress, temperature stress 2. Glycosylation Sugar group 3. Thiol-disulfide modifications Redox Interconversion of thiols and disulfide Redox stress, Structural stress

  6. Why are Thiols and Disulfides important? • Cysteine is often involved in redox reactions where the transfer of electrons proceeds via thiol-disulfide exchange reactions • Heterodisulfide Reductase in methanogens • DsbA: electron exchange across membranes • Other functions of Cys • Cys coordinates transition metals in active sites of electron transfer proteins (ferredoxin) • Because Desulfovibrio vulgaris is a SRB, characterizing protein thiol-disulfide states would help understand mechanisms of energy utilization • How does electron transport move protons across the cell membrane and which (if any) thiol-disulfide exchange reactions are associated with proton generation within the cells? • Any other proteins involved in redox stress and related to metabolism? • Understanding response to stress-- Molecular switches in redox-regulated proteins e.g., disulfide bond formation or glutathionylation during oxidative stress • Thioredoxin and glutaredoxin model systems are involved in Arsenate detoxification, oxidative stress response

  7. SOOH SO3H SOH SH S Role Of Thiol Containing Proteins in Oxygen ‘Detoxification’ (sulfenic acid) (sulfonic acid) Thioredoxin peroxidase (cytoplasmic) S S (sulfinic acid) DsbA (periplasmic) H2O2 Free Thiol H-S-Glutathione(cytoplasmic) S-Glutathione Experimental Approach: Monitor modification of thiol containing proteins during O2 stress using ICAT proteomics.

  8. What Is ICAT And Why It Is Useful For This Purpose ICAT: Isotope Coded Affinity Tag Concept- Isotopic mass difference (9 amu) between the two- Heavy and Light- labels Labels reduced Cys residues in a protein MS analysis and the relative ratios of the two labels correlated with protein abundance Wild Type Sample Stress Sample Denature and reduce the sample Label with Heavy Reagent Label with Light Reagent Pool the two samples Digest the samples Cation Exchange Purification Affinity Purification LC-MS/Ms analysis • ICAT is Cys reactive label so this provides an opportunity to directly investigate PTMs • Use ICAT as a differential trapping technique to detect oxidatively modified proteins in vivo

  9. Δ 4.5 Da doublet Δ 4.5 Da doublet singlet Representative ICAT experiment • Doublet indicates the presence of the peptides in both the proteomes for comparison • Ratios from the doublet give the relative abundance of the protein/peptide • Singlet indicates the presence of the protein/peptide only in one proteome

  10. Experimental protocol outline • Grow DvH in the presence of the stress • Cr stress • Oxygen stress • Sample different time points • Control and variable samples from the same time point • Isolate cells and proteome from the cell culture • Lyse cells and quantitate protein • Label cells with ICAT • Differentially label the two samples to be comapred • Digest and purify the peptides • Affinity tag seleectively purifies the labeled peptides only • LC-MS/MS • Analysis of the peptides

  11. Denature Denature and reduce Label with Heavy reagent Label with Light reagent ICAT Experimental Outline Same sample split in two Differentially label the Cys residues Only free thiols labeled All thiols (free and disulfide) labeled Expected Result: ICAT labeled singlets (present in the D/R sample) vs. doublets (present in both samples) indicate Cys oxidation state

  12. E32 (oxygen stressed) samples were labeled with ICAT T0 c/v 0 min T1 c/v 30 min T2 c/v 60 min T3 c/v 120 min C= control; V= variable (stress) sample • Multiple DvH proteins containing light-ICAT labeled cysteine were observed • Only light label observed in this experimental protocol • Heavy labeled peptides not observed!  Problem with either the protocol or our procedure • Modify the experimental procedure

  13. Modified Experimental ICAT Workflow WT Stressed Denature the samples NEM modification Denature/ reduce +heavy + light • Advantages of this approach: • ICAT labeling proceeds as recommended under denaturing and reducing conditions • Sample complexity is reduced since most of the Cys residues are ‘capped’ • Targeted approach to identify only the Cys residues that are modified with via • disulfide formation or other Cys modification • Regular ICAT done in parallel with the non-NEM modified samples to quantify changes in • protein abundance

  14. Workflow for Cys PTM analysis • Protocol controls and optimization scheme • Use BSA and OVA for labeling and protocol optimization • Use WT DvH samples (WT/WT) for labeling and protocol optimization • DvH Test Case • E111 Cr stress samples: samples stressed with 0.45 mM Cr(VI) • Samples expected to have some modification due to the presence of Cr redox stress • Identify PTMs in O2 stress (E 32) samples • Analyze and identify the Cys modifications • Platform and protocols for H2O2 stress analysis

  15. Growth Curve for E111: Cr(VI) Stress (0.45mM) of D. vulgaris wildtype • An uptick in growth in the variable as compared to the control at 60 minutes

  16. LC/MS/MS Instrumentation and Data Acquisition Eksigent nanoLC2D LCQ Classic ion trap mass spectrometer Data Dependent Acquisition

  17. Raw LC/MS/MS Data to Expression Ratios (Bioworks 3.3) combine “like” MS/MS in the LC/MS/MS file generate peaklist for each resulting MS/MS search each peaklist against DVH protein database with Sequest to match peptide sequence determine H:L ratio for each peptide match with PepQuan

  18. BSA, 1:1 ratio, ICAT

  19. BSA, 1:1 ratio, ICAT

  20. Reverse database searches can be used to estimate match score cutoffs

  21. Measured H:L Ratios BSA, 1:1 mix, ICAT

  22. Measured H:L Ratios Ovalbumin and WT DVH, 1:1 mix, ICAT *std dev = 2.66 with outlier (H:L 109.74) removed

  23. WT DVH, 1:1 mix, NEM/ICAT

  24. ICAT Summary • Most proteins identified in ICAT are the most abundant proteins in DvH • Stress response proteins identified • Potential targets for follow-up analysis • Data mining for protein and peptide expression patterns/ other info

  25. Control protein/peptide example DVU3104: Peptidoglycan associated lipoprotein Related to Omp family of proteins 167 aa MNMFKRVGLVLTLALVLAAGFGCAKKQVAATPEAAPVSDMGDSKSADMAALARAQQIITDAKVYFEFDKFDLKAESKEVLKQKAEVMKKFPSIRVLVEGHCDERGTQEYNLALGERRARA AYEYLVMLGVNAGQLEMISYGKERPAVEGHGEAAWSKNRRDEFKVIK R.VLVEGHCDER.G Peptide identified is underlined in the protein sequence Single peptide that is identified across all conditions and does not show any significant change in either relative abundance or modification

  26. Universal Stress Protein UspA: DVU0261 MFKKIIFATSASPACDNAAKVAFDLARRNKAR ▼LYVFHVLGVPSRGFSQVVRDL▼RTGEEEHHDADYRDWVLEEMKQTYSYQLETYGSPDVVMQVVAGVPHTEIL▼RFARQEG ADLIVMGANTREEDPGASRVRSVIGNTMQRVARSARCPVLIVNRPCTTCWNLFSNIVFSTDFSKAADSAFNFALKTASQIGAKLYLFHACDLAANPMGVFAGQVEIERRIEKA QALMQERYVSKMGDFENYQVDIWEGTPYVEILKYARARQADLIVMAHHTKEGEVEEAEIGSTVEQVVL▼RSACPVASVSKPEKAVV ▼- indicates tryptic digest site, peptide identified is underlined in the protein sequence Peptide: R.SACPVASVSKPEK.A

  27. Universal Stress Protein UspA: DVU0261 MFKKIIFATSASPACDNAAKVAFDLARRNKAR ▼LYVFHVLGVPSRGFSQVVRDL▼RTGEEEHHDADYRDWVLEEMKQTYSYQLETYGSPDVVMQVVAGVPHTEIL▼RFARQEG ADLIVMGANTREEDPGASRVRSVIGNTMQRVARSARCPVLIVNRPCTTCWNLFSNIVFSTDFSKAADSAFNFALKTASQIGAKLYLFHACDLAANPMGVFAGQVEIERRIEKA QALMQERYVSKMGDFENYQVDIWEGTPYVEILKYARARQADLIVMAHHTKEGEVEEAEIGSTVEQVVL▼RSACPVASVSKPEKAVV ▼- indicates tryptic digest site, peptide identified is underlined in the protein sequence Peptide: R.SACPVASVSKPEK.A • - Control: Variable ratios are very consistent across different time points for this peptide which indicates that the peptide (=protein) abundance does not change dramatically. • T2 ratios need to be examined further • ND indicates ratios that could not be determined by the software; will need manual examination

  28. Universal Stress Protein UspA: DVU0261 MFKKIIFATSASPACDNAAKVAFDLARRNKAR ▼LYVFHVLGVPSRGFSQVVRDL▼RTGEEEHHDADYRDWVLEEMKQTYSYQLETYGSPDVVMQVVAGVPHTEIL▼RFARQEG ADLIVMGANTREEDPGASRVRSVIGNTMQRVARSARCPVLIVNRPCTTCWNLFSNIVFSTDFSKAADSAFNFALKTASQIGAKLYLFHACDLAANPMGVFAGQVEIERRIEKA QALMQERYVSKMGDFENYQVDIWEGTPYVEILKYARARQADLIVMAHHTKEGEVEEAEIGSTVEQVVL▼RSACPVASVSKPEKAVV ▼- indicates tryptic digest site, peptide identified is underlined in the protein sequence Peptide: R.SACPVASVSKPEK.A • Control: Variable for NEM blocked samples indicate that the disulfide is present in the control that is either • reduced in the variable samples or reduced and modified in the variable sample- first indication of a PTM in • the protein. • - The trend shifts more towards increase in the PTM with time in the variable sample • - Needs further follow-up and examination

  29. Universal Stress Protein UspA: DVU0261 MFKKIIFATSASPACDNAAKVAFDLARRNKAR ▼LYVFHVLGVPSRGFSQVVRDL▼RTGEEEHHDADYRDWVLEEMKQTYSYQLETYGSPDVVMQVVAGVPHTEIL▼RFARQEG ADLIVMGANTREEDPGASRVRSVIGNTMQRVARSARCPVLIVNRPCTTCWNLFSNIVFSTDFSKAADSAFNFALKTASQIGAKLYLFHACDLAANPMGVFAGQVEIERRIEKA QALMQERYVSKMGDFENYQVDIWEGTPYVEILKYARARQADLIVMAHHTKEGEVEEAEIGSTVEQVVL▼RSACPVASVSKPEKAVV ▼- indicates tryptic digest site, peptide identified is underlined in the protein sequence Peptide: R.SACPVASVSKPEK.A • The results show that there is target for PTM analysis in DvH stressed with Cr • We can target this peptide/protein for data-dependent MS/MS acquisition and analysis

  30. PTM Identification Using ICAT: Progress Summary • Successes: • We have a working protocol for ICAT analysis of Cys modifications • 4 different protocols optimized with BSA but 1 working protocol for DvH • Identified peptides and proteins with modifications with potential role in stress response • Stress Response Protein identified as potentially modified • Data mining for more insight into the metabolic/ sulfate reduction pathway proteins • Peptides with modifications can be identified  we can identify Cys residues with a redox function! • We are identifying not only the protein but the peptide and mapping the reactive Cys • Challenges: • Most abundant proteins may be masking more important modified proteins  need to increase coverage and sensitivity • data mining challenge! These metabolic proteins may also be telling a story • We need to track one peptide across all time points  challenging task for instrumentation • A combination of iTRAQ and ICAT would give us a good coverage of the PTMs

  31. Future Work: Thiol-disulfide PTMs (From GTL Retreat 2006) • Stresses: • Metal stress • Chromium (cell mass available; commencing analysis) • Iron stress? • Oxygen Stress • Nitrate/nitrite stress • H2O2 • Coculture with methanogens • Environmental Isolates - DePue • Mutants: • Fur mutant vs. WT; Fur mutant (control) vs. Fur mutant (Fe stress) • PerR mutant vs. WT; Per mutant (control) vs. Per mutant (H2O2 stress) • Zur mutant- metal stress related • Proposed Mutants • (based on preliminary data and genome analysis) • DsbA (Why?  function in disulfide bond formation) • Thiol:disulfide exchange protein (Why?  thiol-disulfide stress) • Rubredoxin (Why?  electron transfer/ oxygen/ oxidative stress)

  32. Co-culture Proteomics • Commencing work on co-culture proteomics with Alyssa and Aindrila • A combination of ITRAQ and DIGE to investigate co-culture vs. monoculture • iTRAQ to be used as the technique to identify the changes in proteome • DIGE to benchmark the most abundant proteins for comaprison of monoculture to co-culture • Analysis of the most abundant proteins in WT DvH • Fluorescence units for labeled proteins correlated with abundance • We have identified ~60 most abundant proteins in DvH and are analyzing ~ 300 more protein spots identified from DIGE gels

  33. Most abundant proteins in DvH

  34. Most abundant proteins in DvH (contd…)

  35. Co-culture Proteomics • Commencing work on co-culture proteomics with Alyssa and Aindrila • A combination of ITRAQ and DIGE to tease out the data • iTRAQ to be used as the technique to identify the changes in proteome • DIGE to benchmark the most abundant proteins for comaprison of monoculture to co-culture • Analysis of the most abundant proteins in WT DvH • Fluorescence units for labeled proteins correlated with abundance • We have identified ~60 most abundant proteins in DvH and are analyzing ~ 300 more protein spots identified from DIGE gels • Subcellular fractionation to reduce sample complexity and increase coverage • Especially important since co-culture would have ~ 2x as many proteins as monocultures • Membranes would be interesting subsets to analyze

  36. Future Work: Thiol-disulfide PTMs (From GTL Retreat 2006) • Stresses: • Metal stress • Chromium (cell mass available; commencing analysis) • Iron stress? • Oxygen Stress • Nitrate/nitrite stress • H2O2 • Coculture with methanogens • Environmental Isolates - DePue • Mutants: • Fur mutant vs. WT; Fur mutant (control) vs. Fur mutant (Fe stress) • PerR mutant vs. WT; Per mutant (control) vs. Per mutant (H2O2 stress) • Zur mutant- metal stress related • Proposed Mutants • (based on preliminary data and genome analysis) • DsbA (Why?  function in disulfide bond formation) • Thiol:disulfide exchange protein (Why?  thiol-disulfide stress) • Rubredoxin (Why?  electron transfer/ oxygen/ oxidative stress)

  37. Mutants vs. WT analysis: JW 707 Fur Mutant pH10 pH3 High MW Low MW

  38. pH10 pH3 High MW Low MW Mutants vs. WT analysis: JW 708 PerR Mutant

  39. pH10 pH3 High MW Low MW Mutants vs. WT analysis: JW 709 Zur Mutant

  40. Carrie Kozina George Buffleben Arlene Gonzales Gabriela Chirica Acknowledgements Sandia Team: • Our Fearless Leader: Anup Singh • Sara Gaucher LBL: Terry Hazen Lab • Richard Phan • Dominique Joyner

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