# Presentation: 078389

## Detection and Identification of Metabolites from Desulfovibrio Vulgaris Hildenborough Lysate by Capillary Electrophoresis-Electrospray Ionization-Time of Flight-Mass Spectrometry

**Detection and Identification of Metabolites from *****Desulfovibrio Vulgaris***** *****Hildenborough***** Lysate by Capillary Electrophoresis-Electrospray Ionization-Time of Flight-Mass Spectrometry**

- Edward E.K. Baidoo, Peter I. Benke, Francesco Pingitore, Aindrila Mukhopadhyay and Jay D. Keasling.

- The microorganism *Desulfovibrio vulgaris Hildenborough***,*** *because of its metabolic versatility, its ability to remediate heavy metals and radionuclides, coupled with the ease with which it can be maintained in culture is of particular interest to the DOE.** **However, the effective implementation of remediation strategies and the use of natural attenuation for the cleanup of heavy metal waste in DOE sites is dependent upon understanding critical chemical, physical, and biological processes. Thus, an understanding of regulatory mechanisms and cellular responses to different environmental factors affecting the metal remediation activity *in situ* is of great importance. One approach to study such  mechanisms within *D. vulgaris* is to quantify all metabolites within the organism at a given point in time (metabolomics). Since the metabolome is further down the line from gene function, it can reflect more closely the activities of a cell at the functional level than the transcript and the proteome. Capillary electrophoresis and time of flight mass spectrometry (CE-TOFMS) is a promising technique for metabolome research as it provides high separation efficiency and accurate mass determination of compounds with low sample volume requirements. We have utilized novel CE-TOFMS methods for the detection and identification of anionic and cationic metabolites within *D. vulgaris* at mid-log phase. This approach has proven successful in identifying metabolites from most classes of compound, including amino acids, coenzyme As, nucleosides, nucleotides and organic acids.

**Strategy for analysis**

- Pipeline biomass production

**Quenching/Extraction of metabolites**

**Sample clean-up by SPE**

- Proteomics

- Transcriptomics

**Metabolomics**

- Cations

- Anions

- CE+ive
- MS+ive

- CE+ive
- MS-ive

**Separation  of metabolites**

| Compound Name | Molecular formula | Theoretical mass |
| --- | --- | --- |
| Imidazol | C3H4N2 | 68.0374 |
| Glycine | C2H5NO2 | 75.0320 |
| Pyruvic acid | C3H4O3 | 88.0160 |
| Alanine | C3H7NO2 | 89.0477 |
| Sarcosine | C3H7NO2 | 89.0477 |
| Serine | C3H7NO3 | 105.0426 |
| Cytosine | C4H5N3O | 111.0433 |
| Uracil | C4H4N2O2 | 112.0273 |
| Proline | C5H9NO2 | 115.0633 |
| Valine | C5H11NO2 | 117.0790 |
| Betaine | C5H11NO2 | 117.0790 |
| Succinic acid | C4H6O4 | 118.0266 |
| Threonine | C4H9NO3 | 119.0582 |
| Cysteine | C3H7NO2S | 121.0197 |
| 2-Phenylethylamine  | C8H11N | 121.0891 |
| Nicotinamide | C6H6N2O | 122.0480 |
| Nicotinic acid | C6H5NO2 | 123.0320 |
| Aminolevulinic acid | C5H9NO3 | 131.0582 |
| Isoleucine | C6H13NO2 | 131.0946 |
| Leucine | C6H13NO2 | 131.0946 |
| Asparagine | C4H8N2O3 | 132.0535 |
| Ornithine | C5H12N2O2 | 132.0899 |
| Aspartate | C4H7NO4 | 133.0375 |
| Homocysteine | C4H9NO2S | 135.0354 |
| Adenine | C5H5N5 | 135.0545 |
| Hypoxanthine | C5H4N4O | 136.0385 |
| 4-Aminobenzoic acid | C7H7NO2 | 137.0477 |
| Spermidine | C7H19N3 | 145.1579 |
| Lysine | C6H14N2O2 | 146.1055 |
| Glutamate | C5H9NO4 | 147.0532 |
| Methionine | C5H11NO2S | 149.0510 |
| Guanine | C5H5N5O | 151.0494 |
| Histidine | C6H9N3O2 | 155.0695 |
| Orotic acid | C5H4N2O4 | 156.0171 |
| Carnitine  | C7H15NO3 | 161.1052 |
| Hydroxylysine | C6H14N2O3 | 162.1004 |
| Phenylalanine | C9H11NO2 | 165.0790 |
| Pyridoxine | C8H11NO3 | 169.0739 |
| Aconitic acid | C6H6O6 | 174.0164 |
| Arginine | C6H14N4O2 | 174.1117 |
| Citrulline | C6H13N3O3 | 175.0957 |
| Glucosamine  | C6H13NO5 | 179.0794 |
| Tyrosine | C9H11NO3 | 181.0739 |
| Spermine | C10H26N4 | 202.2157 |
| 6-aminopenicillinic acid | C8H12N2O3S | 216.0569 |
| O-succinyl homoserine | C8H13NO6 | 219.0743 |
| N-acetylglucosamine | C8H15NO6 | 221.0899 |
| Cystathionine | C7H14N2O4S | 222.0674 |
| 2-Deoxycytidine | C9H13N3O4 | 227.0906 |
| Cytidine | C9H13N3O5 | 243.0855 |
| DeoxyInosine | C10H12N4O4 | 252.0859 |
| Glucose-6-phosphate | C6H13O9P | 260.0297 |
| 2-Deoxyguanosine | C10H13N5O4 | 267.0968 |
| Inosine | C10H12N4O5 | 268.0808 |
| Arginosuccinic acid | C10H18N4O6 | 290.1226 |
| Uridine-5-monophosphate | C9H13N2O9P | 324.0359 |
| 2-Deoxyguanosine-5-monophosphate | C10H14N5O7P | 347.0631 |
| Adenosine-5-monophosphate | C10H14N5O7P | 347.0631 |
| Guanosine-5-monophosphate | C10H14N5O8P | 363.0580 |
| Xanthosine-5-monophosphate | C10H13N4O9P | 364.0420 |
| Thymidine-5-diphosphate | C10H16N2O11P2 | 402.0229 |
| Cytidine-5-diphosphate | C9H15N3O11P2 | 403.0182 |
| Uridine-5-diphosphate | C9H14N2O12P2 | 404.0022 |
| Adenosine-5-diphosphate | C10H15N5O10P2 | 427.0294 |
| Uridine-5-diphospho-N-acethyl-glucoseamine | C17H27N3O17P2 | 607.0816 |
| Flavin adenine dinucleotide | C27H33N9O15P2 | 785.1571 |
| Malonyl CoA | C24H38N7O19P3S | 853.1156 |

- Table 1. Metabolites Identified from *D. vulgaris* lysate

- Identification of metabolites can be made possible through accurate mass measurements and empirical formula generation. However, when considering structural isomers, accurate mass measurements alone do not provide conclusive identification of metabolites as several compounds can have the same empirical formula and hence the same molecular mass. In such cases the elution order from CE separation is required for identification with a high degree of confidence. This can be obtained by comparing the elution order of the compound of interest with the chemical standard. Such an approach can be referred to as targeted analysis. The metabolites shown in Table 1 were identified using this methodology.
- From the selection of metabolites that we have shown in Table 1 we can gain insights into specific aspects of *D. vulgaris* metabolism. For example, cysteine, serine and homoserine all play a significant role in methionine biosynthesis. Interestingly, O-succinyl homoserine, cystathionine and homocysteine are also intermediates in methionine metabolic pathways.   Furthermore the product of glycolysis, pyruvic acid, as well as citric acid cycle intermediates succinic acid and malonyl coenzyme A, were also identified. Products and intermediates of purine and pyrimidine metabolism, products of the urea cycle, as well as intermediates in secondary metabolism were also observed. We are in the process of obtaining standards for the remaining metabolites of the major *D. vulgaris* pathways in order to characterize metabolism in this organism as fully as possible.
- The 67 metabolites found in *D. vulgaris* lysate (Table 1) represent only a small fraction of the total metabolite pool. This was further emphasized by the much larger number of unknown compounds that were also observed from *D. vulgaris* lysate (Table 2). By obtaining chemical standards we hope to address the identities of these unknown compounds.

|  | Number of metabolites |
| --- | --- |
| D. vulgaris targeted | 67 |
| D. vulgaris unknown | 521 |
| Total | 588 |

- Table 2. Number of metabolites detected from *D. vulgaris* lysate

- Utilize more chemical standards to conduct targeted analysis on a wider pool of metabolites from the various metabolic pathways in order to identify these unknown compounds.
- To apply our current methodology to stress versus control *D. vulgaris* cultures in order to study the response of this organism to various environmental factors.

- Metabolites from most classes of compound, including amino acids, coenzyme As, nucleosides, nucleotides and organic acids were identified by CE-TOFMS.
- A total of 588 metabolites were observed from *D. vulgaris* lysate, of which 67 were targeted.

*D. vulgaris* cells were inoculated from a log phase culture at 10% (v/v). The cell culture was grown in an anaerobic chamber at 30°C to an OD of ~0.3 (~10^8 cells/ml).