# Presentation: 049355

## Proteomic profiling of D. vulgaris in response to Oxygen stress

- Goal
- To identify proteins and the associated pathways in response to different stress
- To compare to the transcriptome data and to understand the regulation of gene expression at both mRNA and protein levels
- Approach
- Whole cell proteomic profiling
- Rapid one-fraction protocol for protein extraction
- 3D LC MS/MS for comprehensive peptide and protein identification
- Data analysis
- Spectra counts for relative protein abundance from replicates
- LPE (local-pooled-error) for statistical evaluation of significant changes

## 3D LC (Verve)TM MS/MS Technology

- 1. Reverse Phase Gradient:

- (0

- -

- X

- n

- %, X

- n

- -

- X

- n+1

- ,...., X

- n+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%)

- Gold lead for liquid

- electrical contact (1.5 kV)

- RP

- SCX

- RP

- Entrance of

- Mass Spectrometer

-  Fully automated

- *patent pending: Verve

- TM

- IEX, 5x

- RP2, 60x

- MS1, 200,000x

- MS2, 200,000x

- C|Y|I|Q|N|C|P|R G-NH2

-  Minimum     Sample Loss

***Wei et al (2005) J. of Proteome Research***

## A summary of the Diversa proteomic experiments

- Protein identification

- Distinct peptide identification

**Salt stress**

**Salt stress**

**Nitrate stress**

**Oxygen stress**
**(E41)**

**Nitrate stress**

**Oxygen stress**
**(E41)**

**C0**

**C1**

**V1**

**C0**

**C1**

**V1**

**C0**

**C1**

**V1**

**C0**

**C1**

**V1**

**C1**

**V1**

**C1**

**V1**

**C0: control at time point 0**
**C1: control at time point 1**
**V1: treatment at time point 1**

## Correlation of proteomic data from different stress experiments

**Cor=0.601**

**Cor=0.648**

- Salt stress, C0-1

- Nitrate stress, C0-1

- Salt stress, C1-1

- Nitrate stress, C1-1

## Summary of E31 Salt Stress

- Expression of proteins involved in a number of metabolic pathways such as energy metabolic pathway, was detected, except for most of the cytochrome C complex
- Not a lot of significant changes were detected upon cell’s exposure to salt stress
- Among proteins which have changed expression following salt stress,
- majority of them have reduced expression
- ABC transporters
- Components in the energy transduction pathway
- Very few of them have induced expression

## Energy transduction and metabolism in Salt Stress

**Complex**

- Energy transduction and metabolism in Salt Stress

- Outside cell

- Periplasm

- Cytoplasm

**H****+**

**H****+**

**ADP + P****i**

**ATP**

**Periplasmic**
**hydrogenase**

- NiFe hydrogenase isozyme 1:** **_**DVU1921-22**_
- NiFeSe hydrogenase:** **_**DVU 1917-18**_

**H****2**

**2H****+**

**2e****-**

**Proton **
**gradient**

**C3**

**C3**

**C3**

**C3**

**Cytochrome C**

**e****-**

**Membrane-bound**
**cytoplasmic**
**hydrogenase**

**cooMKLXUHF: **_**DVU2286-2291**_

**lactate**

**pyruvate**

**acetylCoA**

**acetylphosphate**

**acetate**

**Lactate**
**permease**

_**DVU3026**_

- L-lactate
- dehydrogenase
_**DVU0600**_

- pyruvate
- ferredoxin oxidoreductase
- (**DVU3025 PorB**)

- CoA

**2H****+**** + 2e****-**

**H****2**

**2H****+**** + 2e****-**

- phosphate
- acetyltransferase
- (**DVU3029**)

- acetate kinase
_**DVU3030**_

**TCA cycle**

**SO****4****=**

**APS**

**SO****3****=**

**S****=**** + 2H****+**

**H****2****S**

**2e****-**

**6e****-**

**H****2****S**

- sulfate
- adenylyltransferase
_**DVU1295**_

- adenylyl-sulphate reductase
_**DVU0846-847**_

**?**

- ATP synthase
_**DVU0774-780**_

## Summary of E37 Nitrate stress

-  Down regulated: 30  (C1 vs V1)
-  flagella system
-  ribosomal proteins
-  Up regulated: 75 (C1 vs V1)
- Nitrogen assimilation response regulators
- Chemotaxis related proteins, such as CheY, CheZ
- A number of stress-related proteins,
- universal stress protein family
- desulfoferrodoxin (Rbo)/rubredoxin, rubrerythrin (Rbr)
- hybrid cluster protein
- A number of ABC transporters
- Pathways related to Nitrogen assimilation
- Urea cycle

## The Urea Cycle

**ArgJ**

**ArgB**

**ArgC**

**ArgD**

**ArgJ**

**ArgF**

**ArgG**

**ArgH**

**ProB**

**ProA**

## Transcriptome and Proteomic Data

| C1-1 | C1-2 | V1-1 | V1-2 | #AA | Log (fold-
change) | p-value | Sequence | VIMSS | ORF ID | Log (fold-
change) | Z-score |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 2 | 5 | 6 | 5 | 393 | 0.795193 | 0.218293 | DVU0823 arginine biosynthesis bifunctional protein, ArgJ | 206250 | ORF00299 | -0.711231 | -0.814191 |
| 6 | 7 | 8 | 15 | 308 | 0.759999 | 0.261891 | DVU1466 acetylglutamate kinase, ArgB | 206911 | ORF01358 | -0.49102 | -0.88508 |
| 6 | 1 | 5 | 5 | 354 | 1.032159 | 0.109545 | DVU0492 N-acetyl-gamma-glutamyl-phosphate reductase, ArgC | 209429 | ORF05478 | -0.25646 | -0.354195 |
| 40 | 59 | 80 | 85 | 399 | 0.766086 | 0.016359 | DVU2347 acetylornithine aminotransferase, ArgD | 207835 | ORF02876 | 1.38115 | 1.59966 |
| 15 | 14 | 20 | 17 | 460 | 0.350285 | 0.507071 | DVU1094 argininosuccinate lyase, ArgH | 206529 | ORF00757 | N/A | N/A |
| 50 | 39 | 62 | 81 | 396 | 0.685106 | 0.040818 | DVU1095 argininosuccinate synthase, ArgG | 206530 | ORF00759 | -0.70114 | -1.26679 |
| 3 |   | 12 | 13 | 300 | 6.17486 | 0.124564 | DVU1096 ornithine carbamoyltransferase, ArgF | 206531 | ORF00760 | 1.13479 | 2.07804 |
|   | 2 | 3 |   | 380 | N/A | N/A | DVU0930 glutamate 5-kinase, ProB | 206361 | ORF00482 | 1.3715 | 1.61937 |
| 7 | 11 | 9 | 9 | 419 | 0.039244 | 0.953522 | DVU1953 gamma-glutamyl phosphate reductase, ProA | 207423 | ORF02182 | 0.172361 | 0.334089 |

- Protein

- mRNA

## E41 – The Oxygen Stress Experiment

** **C0

- C1

** ****V0**

**V1**

- T = 0 h

- T = 4 hr

- ctrl

- 1000ppm

- *Numbers of proteins whose expression has changed under two different conditions, identified by LPE
- (*p* <= 0.05)

**72**

## COG functional classification of proteins whose expression changed upon Oxygen stress

## Up-regulated Proteins

| DVU ID | Descriptions | C1-1 | C1-2 | V1-1 | V1-2 | zstats | p-value | ORF ID | Fold change | Z-score |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| DVU2668 | UDP-N-acetylglucosamine pyrophosphorylase, putative | 0 | 0 | 4 | 6 | 2.2598 | 0.0238 | ORF03401 | -0.03687 | -0.05995 |
| DVU0772 | hypothetical protein | 0 | 0 | 5 | 3 | 2.1698 | 0.03 | ORF00213 | 0.209386 | 0.359075 |
| DVU0255 | hypothetical protein | 0 | 0 | 3 | 4 | 2.1251 | 0.0336 | ORF05062 | 0.058507 | 0.093961 |
| DVU2673 | anaerobic glycerol-3-phosphate dehydrogenase, subunit A, truncation | 0 | 0 | 2 | 6 | 2.1251 | 0.0336 | ORF03409 | 0.136531 | 0.254366 |
| DVU0765 | hydroxypyruvate reductase, putative | 0 | 0 | 2 | 3 | 1.9856 | 0.0471 | ORF00198 | 0.108076 | 0.184631 |
| DVU2247 | antioxidant, AhpC/Tsa family | 1 | 1 | 21 | 16 | 2.958 | 0.0031 | ORF02711 | 0.198445 | 0.355907 |
| DVU1851 | peptidase, M23/M37 family | 1 | 1 | 21 | 4 | 2.2264 | 0.026 | ORF02008 | 0.125761 | 0.197134 |
| DVU1898 | glycyl-tRNA synthetase, alpha subunit | 2 | 2 | 4 | 17 | 2.5247 | 0.0116 | ORF02088 | -0.16644 | -0.29439 |
| DVU0766 | transporter, putative (COG2823: Predicted periplasmic or secreted lipoprotein) | 2 | 4 | 13 | 9 | 2.5657 | 0.0103 |  |  |  |
| DVU0665 | nitrogen fixation protein nifU | 2 | 5 | 11 | 12 | 2.532 | 0.0113 |  |  |  |
| DVU1586 | thioredoxin family protein | 2 | 3 | 11 | 7 | 2.3491 | 0.0188 |  |  |  |
| DVU2150 | dnaK suppressor protein, putative | 15 | 19 | 36 | 80 | 3.62 | 0.0003 | ORF02534 | 0.017592 | 0.024513 |
| DVU1869 | methyl-accepting chemotaxis protein | 3 | 4 | 11 | 11 | 2.3134 | 0.0207 | ORF02038 | -0.05802 | -0.10141 |
| DVU1305 | ribosomal protein L23 | 2 | 5 | 24 | 4 | 2.1841 | 0.029 | ORF01096 | -0.10576 | -0.19626 |
| DVU0943 | membrane protein, putative | 22 | 25 | 55 | 80 | 3.5319 | 0.0004 | ORF00506 | -0.14152 | -0.23065 |
| DVU1834 | pyruvate carboxylase, putative | 20 | 7 | 27 | 39 | 2.7008 | 0.0069 | ORF01978 | 0.207468 | 0.368976 |
| DVU0344 | methyl-accepting chemotaxis protein | 5 | 5 | 13 | 14 | 2.1978 | 0.028 | ORF05215 | 0.189834 | 0.344524 |
| DVU1857 | methyl-accepting chemotaxis protein | 3 | 12 | 14 | 15 | 2.0227 | 0.0431 | ORF02017 | 0.112846 | 0.189674 |
| DVU1282 | phosphoglucosamine mutase | 5 | 31 | 24 | 33 | 2.1963 | 0.0281 | ORF01060 | -0.00722 | -0.01307 |
| DVU2325 | mercuric transport protein periplasmic component | 20 | 25 | 45 | 50 | 2.4807 | 0.0131 | ORF02838 | 0.389476 | 0.581495 |
| DVU2105 | hypothetical protein | 24 | 45 | 53 | 91 | 2.6159 | 0.0089 | ORF02450 | 0.601672 | 0.939747 |
| DVU1319 | ribosomal protein L18 | 16 | 21 | 41 | 31 | 2.0358 | 0.0418 |  |  |  |
| DVU2318 | rubrerythrin, putative | 73 | 50 | 91 | 151 | 2.6402 | 0.0083 | ORF02823 | 0.662305 | 1.1359 |
| DVU2771 | hypothetical protein | 25 | 18 | 43 | 37 | 2.0188 | 0.0435 | ORF03572 | 0.240742 | 0.365011 |
| DVU3270 | cytochrome d ubiquinol oxidase, subunit II | 19 | 60 | 73 | 51 | 2.0698 | 0.0385 | ORF04419 | 1.46876 | 2.54238 |
| DVU0386 | amino acid ABC transporter, periplasmic amino acid-binding protein | 74 | 77 | 132 | 129 | 2.2572 | 0.024 | ORF05288 | -0.44892 | -0.77861 |
| DVU2928 | DNA-directed RNA polymerase, beta subunit | 54 | 45 | 113 | 63 | 2.0635 | 0.0391 | ORF03823 | -0.25769 | -0.43344 |
| DVU1228 | thiol peroxidase | 67 | 66 | 126 | 100 | 2.1288 | 0.0333 | ORF00967 | 0.503983 | 0.841449 |

**Protein**

**mRNA**

## Protein 	vs                   mRNA

| DVU ID | C1-1 | C1-2 | V1-1 | V1-2 | Fold-change | p-value | Old ORF IDs | Fold-change | Z-score | Description |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| DVU0779 | 10 | 4 | 7 | 4 | -0.1783 | 0.7261 | ORF00226 | -0.895211 | -1.56913 | ATP synthase F0, B subunit, putative |
| DVUA0116 | 22 | 25 | 20 | 12 | -0.4146 | 0.2143 | ORFA00021 | -0.996692 | -1.32666 | conserved hypothetical protein |
| DVU3026 | 2 | 3 | 1 | 1 | -0.8959 | 0.4380 | ORF03988 | -0.710336 | -1.29346 | L-lactate permease family protein |
| DVU1258 | 7 | 6 | 5 | 12 | 0.1783 | 0.7049 | ORF01019 | -0.746626 | -1.21595 | glutamine synthetase, type III |
| DVU2566 | 15 | 4 | 8 | 3 | -0.4581 | 0.3621 | ORF03241 | -1.08267 | -1.18371 | diaminopimelate decarboxylase |
| DVU2451 | 6 | 4 | 3 | 1 | -1.0397 | 0.1105 | ORF03046 | -0.781389 | -1.17297 | L-lactate permease family protein |
| DVU0693 | 6 | 6 | 3 | 17 | 0.1742 | 0.7198 | ORF00079 | -0.656344 | -1.0723 | molybdopterin oxidoreductase, iron-sulfur cluster-binding subunit, putative |
| DVU2533 | 27 | 27 | 20 | 11 | -0.5990 | 0.0715 | ORF03186 | -0.578177 | -1.06124 | phenylalanyl-tRNA synthetase, beta subunit |
| DVU0671 | 78 | 72 | 70 | 93 | 0.0739 | 0.7576 | ORF00046 | -0.746518 | -1.02451 | conserved hypothetical protein |
| DVU3028 | 29 | 26 | 23 | 25 | -0.1355 | 0.6705 | ORF03992 | -0.564479 | -1.02195 | iron-sulfur cluster-binding protein |
| DVU0918 | 16 | 19 | 21 | 11 | -0.1373 | 0.6959 | ORF00462 | -0.68304 | -1.01488 | ATP synthase F0, A subunit |
| DVU0273 | 23 | 8 | 9 | 6 | -0.6130 | 0.1539 | ORF05098 | 0.594914 | 1.01025 | conserved hypothetical protein |
| DVU3185 | 150 | 144 | 105 | 113 | -0.2996 | 0.1977 | ORF04274 | 0.548165 | 1.0306 | rubredoxin-oxygen oxidoreductase |
| DVU3184 | 7 | 53 | 5 | 31 | -0.4364 | 0.2105 | ORF04273 | 0.649614 | 1.15751 | rubredoxin |
| DVU3183 | 101 | 84 | 101 | 116 | 0.1614 | 0.4937 | ORF04271 | 0.593408 | 1.09403 | desulfoferrodoxin |
| DVU2318 | 73 | 50 | 91 | 151 | 0.6628 | 0.0083 | ORF02823 | 0.662305 | 1.1359 | rubrerythrin, putative |
| DVU0263 | 2 | 3 | 2 | 1 | -0.5493 | 0.4720 | ORF05080 | 0.742436 | 1.08792 | acidic cytochrome c3 |
| DVU3271 | 33 | 36 | 36 | 40 | 0.0962 | 0.7491 | ORF04420 | 0.688061 | 1.20395 | cytochrome d ubiquinol oxidase, subunit I |
| DVU3270 | 19 | 60 | 73 | 51 | 0.5917 | 0.0385 | ORF04419 | 1.46876 | 2.54238 | cytochrome d ubiquinol oxidase, subunit II |
| DVU2411 | 11 | 1 | 2 |   | -3.1550 | 0.2886 | ORF02987 | 0.709336 | 1.21581 | EF hand domain protein |
| DVU3384 | 170 | 203 | 271 | 316 | 0.4544 | 0.0509 | ORF04610 | 0.937757 | 1.24841 | zinc resistance-associated protein |
| DVU2572 |   | 3 | 1 | 3 | 2.3026 | 0.3712 | ORF03251 | 0.718089 | 1.25173 | ferrous iron transport protein A, putative |
| DVU2571 | 3 | 2 | 5 | 1 | -0.0912 | 0.8995 | ORF03249 | 1.34865 | 1.86205 | ferrous iron transport protein B |
| DVU3263 | 3 | 1 | 2 | 2 | 0.1438 | 0.8469 | ORF04404 | 0.735167 | 1.29853 | fumarate reductase, iron-sulfur protein |
| DVU3094 | 651 | 797 | 842 | 875 | 0.1753 | 0.3952 | ORF04114 | 0.78308 | 1.36104 | rubrerythrin |
| DVU2108 | 232 | 207 | 233 | 218 | 0.0280 | 0.9018 | ORF02453 | 1.19499 | 1.36817 | MTH1175-like domain family protein |
| DVU2109 | 776 | 836 | 849 | 877 | 0.0689 | 0.7383 | ORF02456 | 1.44779 | 1.61834 | MTH1175-like domain family protein |
| DVU0036 | 4 | 7 | 13 | 9 | 0.7150 | 0.1148 | ORF04688 | 1.04516 | 1.6872 | hypothetical protein DVU0036 |
| DVU0253 | 51 | 56 | 57 | 58 | 0.0732 | 0.7771 | ORF05060 | 1.0489 | 1.69296 | oxidoreductase, FAD/iron-sulfur cluster-binding domain protein |

**mRNA**
**down**

**mRNA**
**up**

## Expression of Rbr and Rbo  in D. vulgaris

| DVU ID | C1-1 | C1-2 | V1-1 | V1-2 | Log (fold-
change) | p-value | ORF ID | Log (fold-
change) | Z-score | Description |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| DVU3093 |   |   | 1 | 1 |  |  | ORF04112 | 1.1246 | 1.9662 | rubredoxin-like protein  |
| DVU3094 | 651 | 797 | 842 | 875 | 0.17532 | 0.39519 | ORF04114 | 0.7831 | 1.3610 | Rubrerythrin (Rbr) |
| DVU3095 |   |   |   |   |  |  | ORF04117 | 0.5498 | 0.9450 | transcriptional regulator, Fur family |
|  |  |  |  |  |  |  |  |  |  |  |
| DVU2317 | 9 | 8 | 21 | 10 | 0.53522 | 0.19654 | ORF02822 | 0.0091 | 0.0169 | methyl-accepting chemotaxis protein, putative |
| DVU2318 | 73 | 50 | 91 | 151 | 0.66283 | 0.00829 | ORF02823 | 0.6623 | 1.1359 | rubrerythrin, putative |
| DVU2319 |   |   |   |   |  |  | ORF02825 | 0.2655 | 0.4642 |  transcriptional regulator domain protein |
|  |  |  |  |  |  |  |  |  |  |  |
| DVU3183 | 101 | 84 | 101 | 116 | 0.16138 | 0.49371 | ORF04271 | 0.5934 | 1.0940 | Desulfoferrodoxin (Rbo) |
| DVU3184 | 7 | 53 | 5 | 31 | -0.43639 | 0.21046 | ORF04273 | 0.6496 | 1.1575 | rubredoxin |
| DVU3185 | 150 | 144 | 105 | 113 | -0.29955 | 0.19767 | ORF04274 | 0.5482 | 1.0306 | rubredoxin-oxygen oxidoreductase (roO) |

## A comparison between transcriptome data and proteomic data – Oxygen Stress

- mRNA correlation

- protein correlation

- Genes for plotting were selected based  on the following criteria:
-  Avg(Spectrum counts)ctrl or Avg(Spectrum counts)treated >= 5
-  Absolute Z-score between C1 vs V1 >=1
-  Genes were predicted to be in the same operons

- X-axis: operon pair, gene A, log(fold-change)
- Y-axis: operon pair, gene B, log(fold-change)

## A Global comparison - Oxygen stress (E41)

- Genes for plotting were selected based  on the following criteria:
-  Avg(Spectrum counts)ctrl or Avg(Spectrum counts)treated >= 5
-  Absolute Z-score between C1 vs V1 >=1 (for protein)

- X-axis: mRNA expression, log(fold-change)
- Y-axis: protein expression, log(fold-change)

## Comparison of the two Oxygen stress experiments - I

- Very little overlapping between the sets of proteins identified to be up- or down-regulated
- Total number of differentially regulated proteins
- E3: 42 up (ribosomal proteins), 91 down
- Cells dying?
- E41: 28 up (no rubredoxin), 34 down
- Proteins in common:
- Up: DVU2150, dnaK suppressor protein
- Down:
- DVU2981: 2-isopropylmalate synthase (Valine, leucine and isoleucine biosynthesis)
- DVU3274: hypothetical protein, might be an Fe-S cluster as part of an oxidoreductase complex
- DVU2212: COG0314, Molybdopterin converting factor

## E48 Nitrate Stress – long time course

**C**0

**C**1

**C**2

**V**1

**V**2

- S1

- S1

- S2

- S1

- S2

- S1

- S2

- S2

- P1

- P2

- P1

- P2

- P1

- P2

- P2

- P1

- S1

- S2

- P1

- P2

- Analysis in progress

- 4hr

- 4hr

- 8hr

- 8hr

- LC MS/MS:
-  Completed
-  In progress

- Expect LC MS/MS completion ~Sept. 1

## Acknowledgements

- _Proteomics_
- Jun Sun
- Diem Tram
- Jing Wei
- Paul Oeller
- _Management_
- Dennise Wyborski
- Martin Keller
- GTL grant