# Presentation: 819291

## Proteome Analysis of Desulfovibrio vulgaris upon Oxidative Stress Using 3D LC-MS/MS

**Proteome Analysis of *****Desulfovibrio vulgaris***** upon Oxidative Stress Using 3D LC-MS/MS**
**Jing Wei, Wen Yu, Jun Sun, Arianna Jones, Paul Oeller, Jay Short, and Martin Keller**
**Diversa Corporation, 4955 Directors Place, San Diego, CA 92121**

**Oxygen is a common stress factor for anaerobes like *****Desulfovibrio vulgaris***** in the environment. To better understand the cellular responses to such stress, we studied the proteome composition and expression level of *****D. vulgaris***** upon exposing to air.  Whole cell proteomes were extracted from cells before and after sparged with air or N****2****. Protein compositions were assessed using the 3D LC-MS/MS technology developed at Diversa Corporation.  Proteins corresponding to over 50% of the predicted 3674 ORFs were identified among the four samples. Protein abundances in each of the samples were calculated using the sequence coverage of each identified protein. Lists of potential candidates of either up-regulated or down-regulated proteins were obtained. The expressions of some previous studied cytoplasmic oxidative stress protection proteins were also compared. The 3D proteomics technology provided tools for detailed mapping of *****D. vulgaris***** proteome and differential profiling of protein expression in stress response. **

**Abstract**

**Goals**
- Changes in protein levels upon the oxidative stress
**Solutions**
- Rapid one-fraction protocol for protein extraction
- 3D LC MS/MS for comprehensive peptide and protein identification
- Statistical tests based on the duplicated analysis to determine significant changes

**Goals and Solutions**

**Many Proteins exhibited Significant Changes that Coincides with the Oxidative Stress**

**About 50% of the *****D. Vulgaris***** proteome can be assessed for expression changes**
**More than 50 proteins displayed changes upon oxidative Stress**
**Many of these proteins are active involved in the redox processes of the cells**
**Concerted changes in the protein levels observed along the same pathway**

**Conclusions**

**Sample Preparation**

- Culture

- 1% RapiGestTM

- Centrifugation

- Whole cell lysate

- Protease digestion

- Hydrophilic peptides

- Hydrophobic peptides

- 3D LC-MS/MS a

- 3D LC-MS/MS b

**Data**

- 10-20 ml cells

- 350 ug proteins

**Response of Sor Protein **

**2D IEF PAGE**

**Fournier, M.,Zhang, Y.,Wildschut, J.D., Dolla, A., Voordouw, J.K., Schriemer, D.C., Voordouw, G. "Function of Oxygen Resistance Proteins in the Anaerobic, **
**Sulfate-Reducing Bacterium **
***Desulfovibrio vulgaris *****Hildenborough", **
**J.Bacteriol, 2003, 71–79**
** **

**Spot 39 = Sor, **
**superoxide reductase**

**no O****2**** stress**

**1hr O****2**** stress**

**2hr O****2**** stress**

**4hr O****2**** stress**

**Diversa study**

**Sor was also**
**detected in C1V1 & V0V1,**
**down regulated**

**Up-Regulated Proteins (C1/V1)**

- 60 kda chaperonin 1

- chromosome partitioning protein ParB (parB)

- conserved hypothetical protein TIGR01033

-  iojap-related protein

- long-chain fatty acid transport protein, putative

- PAP2 superfamily family

- ribosomal protein L18 (rplR)

- ribosomal protein L21 (rplU)

- ribosomal protein L22 (rplV)

- ribosomal protein L24 (rplX)

- ribosomal protein L3 (rplC)

- ribosomal protein L6 (rplF)

- ribosomal protein L9 (rplI)

- ribosomal protein S1 (rpsA)

- ribosomal protein S16 (rpsP)

- ribosomal protein S20 (rpsT)

- RNA-binding protein

- RNA-binding protein

- DNA-binding protein HU

- spoU rRNA methylase family protein

- sulfate adenylyltransferase (sat)

- translation elongation factor P (efp)

- C1

- V1

- Numbers of Annotated Spectra (median)

- C0/C1 ,C0/V0, V0/V1

*HSP60 homolog, induced by oxygen and heat *

*shock in D. gigas (2D IEF). Also induced by heat *

*shock in D. vulgaris (2D IEF, Sandia)*

**Down-Regulated Proteins (C1/V1)**

- UDP-glucose 6-dehydrogenase (ugd)

- trans-2-enoyl-ACP reductase II (fabK)

- rubredoxin-oxygen oxidoreductase (fprA-2)

- pyruvate flavodoxin/ferredoxin oxidoreductase

- Pyruvate ferredoxin/flavodoxin oxidoreductase

- pyruvate ferredoxin/flavodoxin oxidoreductase

- pyruvate ferredoxin oxidoreductase, alpha subunit

- NADH oxidase, putative

- inosine-5`-monophosphate dehydrogenase (guaB)

- glycolate oxidase iron-sulfur subunit

- ferredoxin I-related protein

- desulfoferrodoxin

- anaerobic ribonucleoside-triphosphate reductase

- aldehyde:ferredoxin oxidoreductase (aor-4)

- iron-sulfur cluster binding protein

- iron-sulfur cluster binding protein

- iron-sulfur cluster binding protein

- GDP-D-mannose dehydratase (gmd)

- delta-aminolevulinic acid dehydratase (hemB)

- 3-isopropylmalate dehydratase, small subunit (leuD)

- V1

- C1

- C0/C1 ,C0/V0, V0/V1

- bacterioferritin (bfr)

- heme biosynthesis protein (nirJ-1)

**Concerted Down Regulation of Proteins along the Polyglucose Utilization Pathway**

- 53, 69, 143, 43,

- ORF03593,

- 940,831,1224,806,

- ORF03986,

- 215,113, 180, 64,

- ORF03994,

- phosphate acetyltransferase

- 346,367,397,446,

- ORF03996,

- acetate kinase

- L-lactate dehydrogenase

- pyruvate ferredoxin oxidoreductase

- 53, 101, 71, 26,

- ORF01978,

- pyruvate carboxylase

- Paula Fareleira, Jean Legall, Anto ́nio v. Xavier and Helena Santos,
- "Pathways for utilization of carbon reserves in *desulfovibrio gigas* under fermentative and respiratory conditions“  *Bacterio*., 1997, 179(12),  3972–3980

**Down-Regulated Proteins (C1/V1)**

- C0/C1 ,C0/V0, V0/V1

- 0.1

- 1

- 10

- 100

- 1000

- universal stress protein family, putative

- TPR Domain domain protein

- thiamine biosynthesis protein ThiC (thiC)

- substrate--CoA ligase

- phosphoribosylformylglycinamidine synthase II (purL)

- phosphate acetyltransferase, putative

- phosphate acetyltransferase (pta)

- perosamine synthetase (lpsC)

- mrp protein (mrp)

- molybdenum cofactor biosynthesis family protein, putative

- Metallo-beta-lactamase superfamily, putative

- isoleucyl-tRNA synthetase (ileS)

- Helicases conserved C-terminal domain protein

- DNA polymerase III, beta subunit (dnaN)

- D-alanine--D-alanine ligase

- ATP-dependent Clp protease, ATP-binding subunit ClpX

- (clpX)

- ATP phosphoribosyltransferase (hisG)

- aspartate kinase, monofunctional class

- adenylosuccinate synthetase (purA)

- adenine deaminase (ade)

- acetyl-CoA synthetase, putative

- acetolactate synthase, putative

- 2-isopropylmalate synthase (leuA)

- C1

- V1

**Acknowledgements**

**The authors would like to thank D.O.E. for the support through the Genome-to-Life Program.**
**Additional support from Diversa Inc. is also acknowledged.**

- Predicted ORFs: **3674**
- Total identified unique proteins: **1766**

**Number of protein identifications**

**1343**

**1338**

**1495**

**1350**

**1060**

**1238**

**1226**

**1140**

**C0**

**V0**

**C1**

**V1**

**# of IDs 1st run**

**# of IDs 2nd run**

**Design of the Oxidative Stress Experiment**

**C0**

**C1**

**V0**

**V1**

**3D LC* Technology**

**0.5 to 1 million MS/MS spectra per injection**
**Minimal sample handling/ SDS tolerance**

*** ****Patent Pending**

- 1. Reverse Phase Gradient:
- 	(0-Xn%, Xn-Xn+1,...., Xn+y-100%)
- 	*i.* salt step (10%, 20%,....90%)
- 	*ii.* wash
- 	*iii*. reverse phase gradient (see step 1)
- 	*iv.* re-equilibrate, go to step *i* (if salt < 100%)
- 2. Back to Step 1. (if salt = 100%)

**C0 **

***vs ***

**V0**

**C0 **

***vs***

** ****C1**

**C1 **

***vs***

** ****V1**

**V0 **

***vs***

** ****V1**

**Lower Cutoff**

**16**

**31**

**81**

**100**

**Higher Cutoff**

**1**

**4**

**45**

**54**

**Origin of the **

**differences**

**Cell growth **

**(small)**

**Cell growth **

**(large)**

**Oxygen stress **

**(large) **

**+                              **

**Cell growth **

**(small)**

**Oxygen stress **

**(large) **

**+                             **

**Cell growth **

**(large)**

- Significant differences

**16**

**31**

**81**

**100**

**1**

**4**

**45**

**54**

- 0

- 20

- 40

- 60

- 80

- 100

- 120

- C0 vs V0

- C0 vs C1

- C1 vs V1

- V0 vs V1

**number of proteins**

- lower cutoff

- higher cutoff