Detection and Identification of Metabolites in Desulfovibrio

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Detection and Identification of Metabolites in Desulfovibrio Powered By Docstoc
					   Detection and Identification of
Metabolites in Desulfovibrio vulgaris
   Hildenborough by CE-TOFMS


           Edward Baidoo
               Contents
•Why is metabolomics necessary?

•Description of our CE-TOFMS strategy
• Results

•Conclusive remarks

•Future work
 Why is metabolomics necessary?

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

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

• 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.
Methionine Metabolism
Structural Diversity of Metabolites
                                                         NH2
                                                                                                                              OH
                                  H
                    H2N           N                                OH                                             O
                                                                                                                          P
                                                                                  N                                                OH

           O                                                            H2N             N                             O            O
                             NH                                O
                                       Arginine            O                                                                  P
H2N
                    OH                                                                                                O            OH
                                                                                                  O
      Glycine                                                      OH         N        N
                                                                                                                                   O
                                                                                                                          P
                                                                                                                  O

                                                          NH2                                                                 OH
                                                                                           HO           OH
                                        N
                                        H                                             Adenosine-5'-triphosphate
                                            Tryptophan




                              O
  O

           OH                                        OH
                     HO

                             Succinic acid
                                                O
      O
                     O
Pyruvic acid

                                               OH
               HO

                             O         O
                         Oxaloacetic acid
                                                                              Acetyl CoA
•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
                                                      Anaerobic




                                                     D. vulgaris culture

Supernatant                                                                Supernatant
      Pellet                                                                     Pellet
                 Centrifuge                                                                  Centrifuge



                                                                                          MeOH        + vortex
           10 % TCA

                                                                                                      Chloroform/ H2O
                                                                                                      +
                                                                                                      vortex
                        + vortex


                        0.5 M tri-n-octyl-amine in

                        Freon
                                                                                            Ultrafiltration
Our Current Approach to Metabolomics

   Transcriptomics         Biomass             Proteomics


                         Metabolomics


             Extraction of metabolites via TCA


                   Sample clean-up by SPE


                   Separation of metabolites

         Cations                            Anions


         CE+ve                             CE+ve
         MS+ve                             MS-ve
 The Need for High Mass Resolution and
Mass Accuracy in Metabolomics Research




               MicrOTOF

           Good mass resolution
           Mass accuracy with confidence
           Ion source compatibility
 An Agilent CE system coupled to a Bruker MicrOTOF




                                           Reflectron



    Nebulizer gas
                         Sheath liquid     Drift region

                         CE sprayer

                   ++
                   +++
                   +++
                    ++
CE inlet

                         Desolvation/Ion                  Orthogonal
           ESI Source                                                  Detector
                         Transfer Optics                  Deflectron
        Which metabolites?

• Targeted

• Non-targeted
    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
                                     Methionine Sulfone (ISD)




                                                            Glutamine

                            Lysine                Valine           Betaine




      Arginine




 15              20   25   30         35             40    Time [min]
    Extracted Ion Mass Electropherogram of Amino acids,
Nucleotides, Organic acids and CoAs via Negative mode CE-MS

                                             Leu                                      Succinic acid
                           AMP
                Arg

                                                         Aspartic acid




0    5     10         15         20     25          30        35         Time [min]
                                      Malonyl CoA
           Propionyl CoA
  Number of Metabolites Observed
 from Desulfovibrio vulgaris Lysate
               Positive Mode   Negative Mode
               CE-TOFMS        CE-TOFMS

Standards            68             64

D. vulgaris
                     37             28
targeted
D. vulgaris
                    409             172
unknown
Total number
                    446             200
observed
  Number of Metabolites Observed
 from Desulfovibrio vulgaris Lysate

                       CE-TOFMS

D. vulgaris targeted      53

D. vulgaris unknown      521

Total number
                         574
observed
         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.
        Acknowledgements
• Jay and Aindrila
• Metabolomics – Peter, Francesco, Sandra
  and David
• Keasling lab
• LBL and UC Berkeley
• DOE