# Presentation: 236753

## 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?

-  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

## 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

## Current Model for Oxygen Detoxification in 

**O****2**

**O****2**

- Periplasm

- Membranes

- Cytoplasm

**O****2**

**O****2**

**H****2****O****2**

**O****2****-****.**

**+ O****2**

**H****2****O****2**

**O****2****-****.**

**H****2****O****2**

**H****2****O**

**H****2****O**

**O****2**

**O****2**

- 1

- 1

- 3

- 4

- 2

- 5

- 6, 7?

- Redox Enzymes
- Superoxide dismutase
- Rubredoxin oxidoreductase

- 4. Rubredoxin
- 5. Cytochrome containing enzymes
- 6. Glutaredoxin; 7. NADH peroxidase

_**Current Model for Oxygen Detoxification in **_
_***Desulfovibrio ***__**species**_

## Post-translational Modifications (PTM) in Stress Response

- 2. Glycosylation

- 3. Thiol-disulfide
-     modifications

- Phosphoryl group

- Sugar group

**Modification**

**Modification Type**

**Common Function**

- Structural stress,
- temperature stress

- Nutrient stress

- Redox Interconversion
- of thiols and disulfide

- Redox stress,
- Structural stress

_**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:**_

## 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

## Role Of Thiol Containing Proteins in Oxygen ‘Detoxification’

**S**

**S**

**S-**Glutathione

**H-S-**Glutathione** **(cytoplasmic)

- DsbA (periplasmic)

- Thioredoxin peroxidase
- (cytoplasmic)

- H2O2

- Free Thiol

- Experimental Approach: Monitor modification of thiol containing proteins during O2 stress
- using ICAT proteomics.

- (sulfinic acid)

- (sulfenic acid)

- (sulfonic acid)

## What Is ICAT And Why It Is Useful For This Purpose

-  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*

- _ICAT_: _I_sotope _C_oded _A_ffinity _T_ag
- 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

- Digest the samples

- Pool the two samples

- Cation Exchange Purification

- Affinity Purification

- LC-MS/Ms analysis

## 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

## 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

## 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

## E32 (oxygen stressed) samples were labeled with ICAT

| DVU0261 | universal stress protein family  |
| --- | --- |
| DVU0402 | dissimilatory sulfite reductase alpha subunit (dsvA)  |
| DVU0566 | GAF domain protein  |
| DVU0607 | adenosylhomocysteinase (ahcY)  |
| DVU0773 | hypothetical protein  |
| DVU0775 | ATP synthase, F1 beta subunit (atpD)  |
| DVU0847 | adenylyl-sulphate reductase, alpha subunit (aprA)  |
| DVU0848 | heterodisulfide reductase, putative  |
| DVU0893 | universal stress protein family  |
| DVU0995 | ThiJ/PfpI family protein  |
| DVU1228 | thiol peroxidase (tpX)  |
| DVU1287 | reductase, iron-sulfur binding subunit, putative  |
| DVU1295 | sulfate adenylyltransferase (sat)  |
| DVU1378 | ketol-acid reductoisomerase (ilvC)  |
| DVU1411 | thiamine biosynthesis protein ThiC (thiC)  |
| DVU1422 | OmpA family protein  |
| DVU2064 | oxidoreductase, 2-nitropropane dioxygenase family  |
| DVU2103 | iron-sulfur cluster-binding/ATPase domain protein  |
| DVU2104 | iron-sulfur cluster-binding/ATPase domain protein  |
| DVU2405 | alcohol dehydrogenase, iron-containing  |
| DVU2538 | threonyl-tRNA synthetase (thrS)  |
| DVU2920 | translation elongation factor Tu (tuf)  |
| DVU3025 | pyruvate-ferredoxin oxidoreductase (poR)  |
| DVU3027 | glycolate oxidase, subunit GlcD (glcD)  |
| DVU3029 | phosphate acetyltransferase (pta)  |
| DVU3065 | AMP-binding enzyme family protein  |
| DVU3104 | peptidoglycan-associated lipoprotein, putative  |
| DVU3183 | desulfoferrodoxin (rbO)  |

- T0 c/v	0 min
- T1 c/v	30 min
- T2 c/v	60 min
- T3 c/v	120 min

- 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

- C= control; V= variable (stress) sample

## Modified Experimental ICAT Workflow

- _WT_

**NEM modification**

- Denature the samples

**+heavy**

- _Stressed_

**+ light**

- Denature/ reduce

_**Modified Experimental ICAT Workflow**_

- _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

## 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 O__2__ stress (E 32) samples_
- Analyze and identify the Cys modifications
- _Platform and protocols for H__2__O__2__ stress analysis_

## Growth Curve for E111: Cr(VI) Stress (0.45mM) of D. vulgaris wildtype  

_**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

## LC/MS/MS Instrumentation and Data Acquisition

- Eksigent nanoLC2D

- LCQ Classic ion trap mass spectrometer

- Data Dependent Acquisition

## Raw LC/MS/MS Data to Expression Ratios

- 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

- (Bioworks 3.3)

## BSA, 1:1 ratio, ICAT

## BSA, 1:1 ratio, ICAT

## Reverse database searches can be used to estimate match score cutoffs

## Measured H:L Ratios

|  | H/L, all | H/L, Ppep =< 10-2 |
| --- | --- | --- |
| avg | 0.93 | 0.92 |
| stddev | 0.26 | 0.13 |

- BSA, 1:1 mix, ICAT

## Measured H:L Ratios

- Ovalbumin and WT DVH, 1:1 mix, ICAT

|  | ova | DVH, all | DVH, P(pep) =< 10-2 |
| --- | --- | --- | --- |
| avg | 1.21 | 1.90 | 1.05 |
| stddev | 0.62 | 5.01* | 0.38 |

- *std dev = 2.66 with outlier (H:L 109.74)  removed

## WT DVH, 1:1 mix, NEM/ICAT

|  | DVH, P(pep) =< 10-2  |
| --- | --- |
| average | 0.84 |
| std dev | 0.38 |

## ICAT Summary

|  | Control:Variable | Control:Variable (NEM) |
| --- | --- | --- |
| Total proteins identified (proteins present in any of the time points) | 101 | 100 |
| Proteins common across all time points (T0-T1-T2-T3-T4) | 13 | 11 |
| Peptides common across all time points (T0-T1-T2-T3-T4) | 2 (!) | 2 (!) |

-  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

## Control protein/peptide example

|  | Control:Variable
(H:L) | Control:Variable (NEM)
H:L |
| --- | --- | --- |
| T-0 | 1.7:1 | 1.8:1 |
| T-1 |    1:1 | 1.3:1 |
| T-2 |       1:1.6 | 1.2:1 |
| T-3 | 1.2:1 | 1.5:1 |
| T-4 |      1:1.5 | 2.0:1 |

*DVU3104: Peptidoglycan associated lipoprotein*
*Related to Omp family of proteins*
*167 aa*
*MNMFKRVGLVLTLALVLAAGFG**C**AKKQVAATPEAAPVSDMGDSKSADMAALARAQQIITDAKVYFEFDKFDLKAESKEVLKQKAEVMKKFPSI*_*RVLVEGH*__*C*__*DERG*_*TQEYNLALGERRARA 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

## Universal Stress Protein UspA: DVU0261

*Peptide: R.SA**C**PVASVSKPEK.A *

|  | Control:Variable
(H:L) | Control:Variable (NEM)
(H:L) |
| --- | --- | --- |
| T-0 | ND | ND |
| T-1 (30min) | 1:2 | 2.9:1 |
| T-2 (60 min) |   1:5.5 | ND |
| T-3 (120 min) | 1:1 |   3.4:1 |
| T-4 (240 min) | ND | 15.5:1 |

*MFKKIIFATSASPA****C****DNAAKVAFDLARRNKAR ***▼***LYVFHVLGVPSRGFSQVVRDL***▼***RTGEEEHHDADYRDWVLEEMKQTYSYQLETYGSPDVVMQVVAGVPHTEIL***▼***RFARQEG*
*ADLIVMGANTREEDPGASRVRSVIGNTMQRVARSAR****C****PVLIVNRP****C****TT****C****WNLFSNIVFSTDFSKAADSAFNFALKTASQIGAKLYLFHA****C****DLAANPMGVFAGQVEIERRIEKA*
*QALMQERYVSKMGDFENYQVDIWEGTPYVEILKYARARQADLIVMAHHTKEGEVEEAEIGSTVEQVVL***▼***R*_*SA*__***C***__*PVASVSKPEKAVV*_* *
**▼**- indicates tryptic digest site, peptide identified is underlined in the protein sequence

## Universal Stress Protein UspA: DVU0261

*Peptide: R.SA**C**PVASVSKPEK.A *

|  | Control:Variable
(H:L) | Control:Variable (NEM)
(H:L) |
| --- | --- | --- |
| T-0 | ND | ND |
| T-1 (30min) | 1:2 | 2.9:1 |
| T-2 (60 min) |   1:5.5 | ND |
| T-3 (120 min) | 1:1 |   3.4:1 |
| T-4 (240 min) | ND | 15.5:1 |

- - 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

*MFKKIIFATSASPA****C****DNAAKVAFDLARRNKAR ***▼***LYVFHVLGVPSRGFSQVVRDL***▼***RTGEEEHHDADYRDWVLEEMKQTYSYQLETYGSPDVVMQVVAGVPHTEIL***▼***RFARQEG*
*ADLIVMGANTREEDPGASRVRSVIGNTMQRVARSAR****C****PVLIVNRP****C****TT****C****WNLFSNIVFSTDFSKAADSAFNFALKTASQIGAKLYLFHA****C****DLAANPMGVFAGQVEIERRIEKA*
*QALMQERYVSKMGDFENYQVDIWEGTPYVEILKYARARQADLIVMAHHTKEGEVEEAEIGSTVEQVVL***▼***R*_*SA*__***C***__*PVASVSKPEKAVV*_* *
**▼**- indicates tryptic digest site, peptide identified is underlined in the protein sequence

## Universal Stress Protein UspA: DVU0261

*Peptide: R.SA**C**PVASVSKPEK.A *

|  | Control:Variable
(H:L) | Control:Variable (NEM)
(H:L) |
| --- | --- | --- |
| T-0 | ND | ND |
| T-1 (30min) | 1:2 | 2.9:1 |
| T-2 (60 min) |   1:5.5 | ND |
| T-3 (120 min) | 1:1 |   3.4:1 |
| T-4 (240 min) | ND | 15.5:1 |

-  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

*MFKKIIFATSASPA****C****DNAAKVAFDLARRNKAR ***▼***LYVFHVLGVPSRGFSQVVRDL***▼***RTGEEEHHDADYRDWVLEEMKQTYSYQLETYGSPDVVMQVVAGVPHTEIL***▼***RFARQEG*
*ADLIVMGANTREEDPGASRVRSVIGNTMQRVARSAR****C****PVLIVNRP****C****TT****C****WNLFSNIVFSTDFSKAADSAFNFALKTASQIGAKLYLFHA****C****DLAANPMGVFAGQVEIERRIEKA*
*QALMQERYVSKMGDFENYQVDIWEGTPYVEILKYARARQADLIVMAHHTKEGEVEEAEIGSTVEQVVL***▼***R*_*SA*__***C***__*PVASVSKPEKAVV*_* *
**▼**- indicates tryptic digest site, peptide identified is underlined in the protein sequence

## Universal Stress Protein UspA: DVU0261

*Peptide: R.SA**C**PVASVSKPEK.A *

|  | Control:Variable
(H:L) | Control:Variable (NEM)
(H:L) |
| --- | --- | --- |
| T-0 | ND | ND |
| T-1 (30min) | 1:2 | 2.9:1 |
| T-2 (60 min) |   1:5.5 | ND |
| T-3 (120 min) | 1:1 |   3.4:1 |
| T-4 (240 min) | ND | 15.5:1 |

-  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

*MFKKIIFATSASPA****C****DNAAKVAFDLARRNKAR ***▼***LYVFHVLGVPSRGFSQVVRDL***▼***RTGEEEHHDADYRDWVLEEMKQTYSYQLETYGSPDVVMQVVAGVPHTEIL***▼***RFARQEG*
*ADLIVMGANTREEDPGASRVRSVIGNTMQRVARSAR****C****PVLIVNRP****C****TT****C****WNLFSNIVFSTDFSKAADSAFNFALKTASQIGAKLYLFHA****C****DLAANPMGVFAGQVEIERRIEKA*
*QALMQERYVSKMGDFENYQVDIWEGTPYVEILKYARARQADLIVMAHHTKEGEVEEAEIGSTVEQVVL***▼***R*_*SA*__***C***__*PVASVSKPEKAVV*_* *
**▼**- indicates tryptic digest site, peptide identified is underlined in the protein sequence

## 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

## 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)

## 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

## Most abundant proteins in DvH

| Accession | Description | Max Score |
| --- | --- | --- |
| DVU2405 | alcohol dehydrogenase, iron-containing {Desulfovibrio vulgaris Hildenborough} | 1180 |
| DVU1295 | sulfate adenylyltransferase (sat) {Desulfovibrio vulgaris Hildenborough} | 1156 |
| DVU1422 | OmpA family protein {Desulfovibrio vulgaris Hildenborough} | 1049 |
| DVU1378 | ketol-acid reductoisomerase (ilvC) {Desulfovibrio vulgaris Hildenborough} | 987 |
| DVU1636 | inorganic pyrophosphatase, manganese-dependent (ppaC) {Desulfovibrio vulgaris Hildenborough} | 969 |
| DVU0873 | translation elongation factor Ts (tsf) {Desulfovibrio vulgaris Hildenborough} | 968 |
| DVU0777 | ATP synthase, F1 alpha subunit (atpA) {Desulfovibrio vulgaris Hildenborough} | 953 |
| DVU1468 | peptidase/PDZ domain protein {Desulfovibrio vulgaris Hildenborough} | 943 |
| DVU3185 | rubredoxin-oxygen oxidoreductase (roO) {Desulfovibrio vulgaris Hildenborough} | 943 |
| DVU2449 | S-adenosylmethionine synthetase (metK) {Desulfovibrio vulgaris Hildenborough} | 933 |
| DVU0403 | dissimilatory sulfite reductase beta subunit (dvsB) {Desulfovibrio vulgaris Hildenborough} | 901 |
| DVU1334 | trigger factor (tig) {Desulfovibrio vulgaris Hildenborough} | 869 |
| DVU0851 | hypothetical protein {Desulfovibrio vulgaris Hildenborough} | 847 |
| DVU1469 | ribosomal protein S1, putative {Desulfovibrio vulgaris Hildenborough} | 830 |
| DVU2399 | hydrogenase, putative {Desulfovibrio vulgaris Hildenborough} | 784 |
| DVU2784 | dehydrogenase, FMN-dependent family {Desulfovibrio vulgaris Hildenborough} | 753 |
| DVU0566 | GAF domain protein {Desulfovibrio vulgaris Hildenborough} | 714 |
| DVU1937 | phosphonate ABC transporter, periplasmic phosphonate-binding protein, putative {Desulfovibrio vulga | 689 |
| DVU3048 | aspartate-semialdehyde dehydrogenase (asd) {Desulfovibrio vulgaris Hildenborough} | 676 |
| DVU0775 | ATP synthase, F1 beta subunit (atpD) {Desulfovibrio vulgaris Hildenborough} | 662 |
| DVU1238 | amino acid ABC transporter, periplasmic amino acid-binding protein {Desulfovibrio vulgaris Hildenbo | 657 |
| DVU0752 | amino acid ABC transporter, amino acid-binding protein {Desulfovibrio vulgaris Hildenborough} | 648 |
| DVU0675 | amino acid ABC transporter, periplasmic amino acid-binding protein {Desulfovibrio vulgaris Hildenbo | 628 |
| DVU0797 | conserved hypothetical protein {Desulfovibrio vulgaris Hildenborough} | 625 |

## Most abundant proteins in DvH (contd...)

| Description | Max Score |
| --- | --- |
| glycine/betaine/L-proline ABC transporter, periplasmic-binding protein {Desulfovibrio vulgaris Hild | 611 |
| MTH1175-like domain family protein {Desulfovibrio vulgaris Hildenborough} | 585 |
| oligopeptide/dipeptide ABC transporter, periplasmic oligopeptide/dipeptide-binding protein {Desulfo | 578 |
| ribosomal protein L1 (rplA) {Desulfovibrio vulgaris Hildenborough} | 571 |
| conserved hypothetical protein {Desulfovibrio vulgaris Hildenborough} | 569 |
| iron-sulfur cluster-binding/ATPase domain protein {Desulfovibrio vulgaris Hildenborough} | 546 |
| adenylate kinase (adk) {Desulfovibrio vulgaris Hildenborough} | 545 |
| ABC transporter, periplasmic substrate-binding protein {Desulfovibrio vulgaris Hildenborough} | 523 |
| TPR domain protein {Desulfovibrio vulgaris Hildenborough} | 520 |
| glutamine ABC transporter, periplasmic glutamine-binding protein (glnH) {Desulfovibrio vulgaris Hil | 516 |
| iron-sulfur cluster-binding/ATPase domain protein {Desulfovibrio vulgaris Hildenborough} | 515 |
| translation elongation factor Tu (tuf) {Desulfovibrio vulgaris Hildenborough} | 501 |
| conserved hypothetical protein {Desulfovibrio vulgaris Hildenborough} | 495 |
| nigerythrin (ngr) {Desulfovibrio vulgaris Hildenborough} | 486 |
| dissimilatory sulfite reductase alpha subunit (dsvA) {Desulfovibrio vulgaris Hildenborough} | 485 |
| polyamine ABC transporter, periplasmic polyamine-binding protein {Desulfovibrio vulgaris Hildenboro | 464 |
| ribosomal protein L25 (rplY) {Desulfovibrio vulgaris Hildenborough} | 411 |
| enolase (eno) {Desulfovibrio vulgaris Hildenborough} | 406 |
| conserved hypothetical protein {Desulfovibrio vulgaris Hildenborough} | 370 |
| ThiJ/PfpI family protein {Desulfovibrio vulgaris Hildenborough} | 347 |
| adenosylhomocysteinase (ahcY) {Desulfovibrio vulgaris Hildenborough} | 343 |
| alcohol dehydrogenase, iron-containing {Desulfovibrio vulgaris Hildenborough} | 308 |
| ribosomal protein L4 (rplD) {Desulfovibrio vulgaris Hildenborough} | 288 |
| rubrerythrin (rr) {Desulfovibrio vulgaris Hildenborough} | 275 |

## 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

## 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)

## Mutants vs. WT analysis: JW 707 Fur Mutant

| Upregulated | Fur Mutant  | Wild Type |
| --- | --- | --- |
| > 1.5 fold | 31 | 33 |
| > 2.0 fold | 6 | 15 |

- pH3

- pH10

- High MW

- Low MW

## Mutants vs. WT analysis: JW 708 PerR Mutant

| Upregulated | PerR Mutant  | Wild Type |
| --- | --- | --- |
| > 1.5 fold | 20 | 11 |
| > 2.0 fold | 8 | 5 |

## Mutants vs. WT analysis: JW 709 Zur Mutant

| Upregulated | Zur Mutant  | Wild Type |
| --- | --- | --- |
| > 1.5 fold | 27 | 26 |
| > 2.0 fold | 15 | 12 |

## Acknowledgements

- 	 _**Sandia Team:**_
- Our Fearless Leader: Anup Singh
- Sara Gaucher

_**LBL: Terry Hazen Lab**_
- Richard Phan
- Dominique Joyner