# Presentation: 078390

## Detection and Identification of Metabolites in Desulfovibrio vulgaris Hildenborough by CE-TOFMS 

- Edward Baidoo

## Contents

- Description of our CE-TOFMS strategy

- Conclusive remarks

- Future work

- Why is metabolomics necessary?

-  Results

## Why is metabolomics necessary?

- A metabolite may come from more than one metabolic pathway and it is only when you conduct a study on the metabolome as a whole that you can identify which pathways are involved in it’s metabolism.

- Metabolomics can be viewed as complementary to transcriptomics and proteomics.

- The metabolome is further down the line from gene function and so reflects  more closely the activities of a cell at the functional level.

- The proteome cannot be completely predicted from the  transcriptome due to some differences in regulatory mechanisms.

## Methionine Metabolism

## Structural Diversity of Metabolites

## Successful extraction of metabolites from cellular matrices is dependent upon the type extraction procedure. 

- When you have a structural diverse group of compounds, it becomes difficult to develop methods that can separate them all.

- Ionization/Visualization of all metabolites is also difficult to achieve, as each compound will vary in its affinity for a detector.

## Extraction

*D. vulgaris* culture

- 10 % TCA

- MeOH

- Ultrafiltration

- Chloroform/ H2O +
- vortex

- 0.5 M tri-n-octyl-amine in Freon

- + vortex

- Anaerobic

## Our Current Approach to Metabolomics

**Our Current Approach to Metabolomics**

- Biomass

- Extraction of metabolites via TCA

- Sample clean-up by SPE

- Proteomics

- Transcriptomics

- Metabolomics

- Cations

- Anions

- CE+ve
- MS+ve

- CE+ve
- MS-ve

- Separation  of metabolites

## The Need for High Mass Resolution and Mass Accuracy in Metabolomics Research

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**MicrOTOF**

**The Need for High Mass Resolution and Mass Accuracy in Metabolomics Research**

- Mass accuracy with confidence

- Good mass resolution

- Ion source compatibility

## An Agilent CE system coupled to a Bruker MicrOTOF 

- CE inlet

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- Sheath liquid

- Nebulizer gas

- CE sprayer

**An Agilent CE system coupled to a Bruker MicrOTOF **

- Desolvation/Ion
- Transfer Optics

- Orthogonal
- Deflectron

- Detector

- Drift region

- Reflectron

- ESI Source

**Notes:**
Ions enter the mass spec/glass capillary via electrospray ionisation and are then focused/channeled through to the orthogonal deflectron, where ions are deflected via an applied potential to the reflectron region, where they are relected back towards the detector. Note the reflectron increases the path length fro separation and hence resolving power. Since the principle of ion separation is through time of flight it is essential that the flight tube does not expand or contract upon temperature fluctuations as this will effect mass accuracies and resolving power. Ideally the TOF requires a < 1C/hr change for optimal performance. Since this is clearly not the case in room 272 we have to use internal calibrants (which are infused through the sheath) to ensure mass accuracies < 5ppm. Thus the TOF can be calibrated at all times during a run cycle. This in itself is difficult as each chemical will have various impurities, some of which are responsible for ion suppression effects (i.e. a reduction in signal to noise of our metabolites).

## Which metabolites?

- Targeted
- Non-targeted

## Slide 12

## Targeted approach requires standards

- 132 compounds:
- 		Amino acids
- 		Purines/ Pyrimidines
- 		Nucleosides
- 		Nucleotides
- 		Co-As
- 		Organic acids

## Identified Standards

| Anionic Species | Theoretical m/z | Measured m/z |   | Cationic Species | Theoretical m/z | Measured m/z |
| --- | --- | --- | --- | --- | --- | --- |
| DL-a-e-Diaminopimelic acid | 189.08808 | 189.087745 |   | Imidazol | 69.044725 | 69.045137 |
| iso-Citric acid | 191.019726 | 191.019767 |   | Glycine | 76.039305 | 76.039759 |
| Ribose-5-phosphate disodium salt | 229.011877 | 229.012029 |   | Alanine | 90.054955 | 90.055493 |
| Ribose-5-phosphate disodium salt | 229.011877 | 229.011911 |   | Sarcosine | 90.054955 | 90.055493 |
| deoxyInosine | 251.078578 | 251.077852 |   | Serine | 106.049870 | 106.050503 |
| Glucose-6-phosphate | 259.022442 | 259.022088 |   | Cytosine | 112.050538 | 112.051207 |
| Adenosine | 266.089477 | 266.089213 |   | Uracil | 113.034554 | 113.035229 |
| 2-deoxyGuanosine monohydrate | 266.089477 | 266.089213 |   | Proline | 116.070605 | 116.071298 |
| Inosine | 267.073493 | 267.07305 |   | Valine | 118.086255 | 118.086960 |
| Arginosuccinic acid | 289.115358 | 289.116405 |   | Betaine | 118.086255 | 118.086960 |
| 2-deoxyCytidine-5-monophosphate | 306.04966 | 306.049033 |   | Threonine | 120.065520 | 120.066237 |
| 2-deoxyUridine -5-monophosphate | 307.033675 | 307.034183 |   | Cysteine | 122.027026 | 122.027755 |
| N-acetyl neuramic acid | 308.098705 | 308.098979 |   | 2-Phenylethylamine | 122.096426 | 122.097155 |
| Thymidine-5- monophosphate | 321.049325 | 321.049203 |   | Nicotinamide | 123.055289 | 123.056024 |
| Thymidine-5- monophosphate | 321.049325 | 321.049166 |   | Nicotinic acid | 124.039305 | 124.040046 |
| Cyttidine-5-monophosphate | 322.044574 | 322.044443 |   | 2-Picolinic acid | 124.039305 | 124.040046 |
| Uridine -5-monophosphate | 323.02859 | 323.028773 |   | Aminolevulinic acid | 132.065520 | 132.066308 |
| 2-deoxyAdenosine-5-monophosphate | 330.060893 | 330.061054 |   | Isoleucine | 132.101905 | 132.102694 |
| 2-deoxyAdenosine-5-monophosphate | 330.060893 | 330.060348 |   | Leucine | 132.101905 | 132.102694 |
| 2-deoxyGuanosine-5-monophosphate | 346.055808 | 346.05579 |   | Asparagine | 133.060769 | 133.061563 |
| Adenosine-5-monophosphate | 346.055808 | 346.055538 |   | L-Ornithine | 133.097154 | 133.097949 |
| Inosine-5-monophosphate | 347.039823 | 347.039666 |   | Aspartate | 134.044784 | 134.045584 |
| Guanosine-5-monophosphate | 362.050722 | 362.051127 |   | DL-Homocysteine | 136.042676 | 136.043488 |
| Xanthosine-5-monophosphate | 363.034738 | 363.034938 |   | Adenine | 136.061772 | 136.062584 |

## Identified Standards

| Anionic species | Theoretical m/z | Measured m/z |   | Cationic Species | Theoretical m/z | Measured m/z |
| --- | --- | --- | --- | --- | --- | --- |
| 2-deoxyCytidine-5-diphosphate | 386.01599 | 386.016165 |   | Hypoxanthine | 137.045787 | 137.046605 |
| Thymidine-5-diphosphate | 401.015656 | 401.015864 |   | 4-Aminobenzoic acid | 138.054955 | 138.055779 |
| Cytidine-5-diphosphate | 402.010905 | 402.011076 |   | Spermidine | 146.165174 | 146.166047 |
| Uridine-5-diphosphate | 402.99492 | 402.995246 |   | Glutamine | 147.076419 | 147.077297 |
| 2-deoxyGuanosine-5-diphosphate | 426.022138 | 426.022387 |   | Lysine | 147.112804 | 147.113682 |
| Adenosine-5-diphosphate | 426.022138 | 426.022268 |   | Glutamate | 148.060434 | 148.061318 |
| Guanosine-5-diphosphate | 442.017053 | 442.016943 |   | O-Acetyl-Serine | 148.060434 | 148.061318 |
| 2-deoxyCytidine-5-triphosphate | 465.982321 | 465.98204 |   | Methionine | 150.058326 | 150.059222 |
| 2-deoxyUridine-5-triphosphate | 466.966336 | 466.966177 |   | Guanine | 152.056686 | 152.057594 |
| Thymidine-5-triphosphate | 480.981986 | 480.981161 |   | Histidine | 156.076753 | 156.077685 |
| Cytidine-5-triphosphate | 481.977235 | 481.977212 |   | L-Carnitine | 162.112470 | 162.113438 |
| Uridine-5-triphosphate | 482.961251 | 482.961099 |   | d-Hydroxylysine | 163.107719 | 163.108693 |
| 2-deoxyAdenosine-5-triphosphate | 489.993554 | 489.993374 |   | Phenylalanine | 166.086255 | 166.087247 |
| 2-deoxyGuanosine-5-triphosphate | 505.988469 | 505.988496 |   | Pyridoxamine | 169.097154 | 169.098164 |
| Adenosine-5-triphosphate | 505.988469 | 505.9879993 |   | Pyridoxine | 170.081170 | 170.082185 |
| Guanosine-5-triphosphate | 521.983383 | 521.98294 |   | Arginine | 175.118952 | 175.119997 |
| Uridine-5-diphosphate galactose | 565.047744 | 565.047728 |   | L-Citrulline | 176.102968 | 176.104019 |
| Guanosine-5-diphosphate glucose | 604.069876 | 604.06952 |   | D(+)-Glucosamine | 180.086649 | 180.087724 |
| Uridine-5-diphospho-N-acethyl-glucoseamine | 606.074293 | 606.074632 |   | Tyrosine | 182.081170 | 182.082257 |
|  |  |  |  | DL-a-e-Diaminopimelic acid | 191.102633 | 191.103774 |
|  |  |  |  | Spermine | 203.223023 | 203.224236 |
|  |  |  |  | Indol-3-pyruvic acid | 204.065520 | 204.066738 |
|  |  |  |  | Tryptophan | 205.097154 | 205.098378 |

## Extracted Ion Mass Electropherogram of Desulfovibrio vulgaris        via CE-TOFMS

**Extracted Ion Mass Electropherogram of *****Desulfovibrio vulgaris*****        via CE-TOFMS**

**Methionine Sulfone (ISD)**

- Valine

- Betaine

- Glutamine

- Lysine

- Arginine

## Extracted Ion Mass Electropherogram of Amino acids, Nucleotides, Organic acids and CoAs via Negative mode CE-MS

**Extracted Ion Mass Electropherogram of Amino acids, Nucleotides, Organic acids and CoAs via Negative mode CE-MS**

- Malonyl CoA

- Propionyl CoA

- Succinic acid

- Leu

- Arg

- AMP

- Aspartic acid

## Number of Metabolites Observed from Desulfovibrio vulgaris Lysate 

|  | Positive Mode
CE-TOFMS | Negative Mode
CE-TOFMS |
| --- | --- | --- |
| Standards | 68 | 64 |
| D. vulgaris targeted | 37 | 28 |
| D. vulgaris unknown | 409 | 172 |
| Total number observed | 446 | 200 |

## Number of Metabolites Observed from Desulfovibrio vulgaris Lysate

|  | CE-TOFMS |
| --- | --- |
| D. vulgaris targeted | 53 |
| D. vulgaris unknown | 521 |
| Total number observed | 574 |

## Conclusions

- Cationic and anionic metabolites were identified by CE-TOFMS.
- A total of 574 metabolites were observed in *D. vulgaris* lysate, of which 53 were targeted.

## Future work

- Target a wider pool of metabolites via CE-TOFMS and CE-FTMS
- Non-targeted approach - Transfer our CE methodologies to the FTMS.
- Utilize Metabolite Tools (Bruker Daltonics)
- 	to expedite future stress versus control experiments.

## Slide 22

## Acknowledgements

- Jay and Aindrila
- Metabolomics – Peter, Francesco, Sandra and David
- Keasling lab
- LBL and UC Berkeley
- DOE