Skip to main content
SpringerLink
Log in
Book cover

Functional Genomics pp 377–398Cite as

Metabolome Analysis of Gram-Positive Bacteria such as Staphylococcus aureus by GC-MS and LC-MS

  • Protocol
  • First Online:

Part of the book series: Methods in Molecular Biology ((MIMB,volume 815))

Abstract

The field of metabolomics has become increasingly important in the context of functional genomics. Together with other ”omics“ data, the investigation of the metabolome is an essential part of systems biology. Beside the analysis of human and animal biofluids, the investigation of the microbial physiology by methods of metabolomics has gained increased attention. For example, the analysis of metabolic processes during growth or virulence factor expression is crucially important to understand pathogenesis of bacteria. Common bioanalytical techniques for metabolome analysis include liquid and gas chromatographic methods coupled to mass spectrometry (LC-MS and GC-MS) and spectroscopic approaches such as NMR. In order to achieve metabolome data representing the physiological status of a microorganism, well-verified protocols for sampling and analysis are necessary. This chapter presents a detailed protocol for metabolome analysis of the Gram-positive bacterium Staphylococcus aureus. A detailed manual for cell sampling and metabolite extraction is given, followed by the description of the analytical procedures GC-MS and LC-MS. The advantages and limitations of each experimental setup are discussed. Here, a guideline specified for S. aureus metabolomics and information for important protocol steps are presented, to avoid common pitfalls in microbial metabolome analysis.

This is a preview of subscription content, log in via an institution.

Protocol
USD   49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD   84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD   159.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD   109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Learn about institutional subscriptions

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more

References

  1. Fiehn O, (2002) Metabolomics – the link between genotypes and phenotypes. Plant Mol Biol 48, 155–171.

    Article  PubMed  CAS  Google Scholar 

  2. Oliver SG, Winson MK, Kell DB, and Baganz F (1998) Systematic functional analysis of the yeast genome. Trends in biotechnology 16, 373–378.

    Article  PubMed  CAS  Google Scholar 

  3. Bennett BD, Kimball EH, Gao M, Osterhout R, Van Dien SJ, and Rabinowitz JD (2009) Absolute metabolite concentrations and implied enzyme active site occupancy in Escherichia coli. Nat Chem Biol 5, 593–599.

    Article  PubMed  CAS  Google Scholar 

  4. Brauer MJ, Yuan J, Bennett BD, Lu WY, Kimball E, Botstein D, and Rabinowitz JD (2006) Conservation of the metabolomic response to starvation across two divergent microbes. P Natl Acad Sci USA 103, 19302–19307.

    Article  CAS  Google Scholar 

  5. Zamboni N, and Sauer U (2009) Novel biological insights through metabolomics and 13C-flux analysis. Curr Opin Microbiol 12, 553–558.

    Article  PubMed  CAS  Google Scholar 

  6. Nakahigashi K, Toya Y, Ishii N, Soga T, Hasegawa M, Watanabe H, Takai Y, Honma M, Mori H, and Tomita M (2009) Systematic phenome analysis of Escherichia coli multiple-knockout mutants reveals hidden reactions in central carbon metabolism. Mol Syst Biol 5, 306.

    Article  PubMed  Google Scholar 

  7. Mashego MR, Rumbold K, De Mey M, Vandamme E, Soetaert W, and Heijnen JJ (2007) Microbial metabolomics: past, present and future methodologies. Biotechnol Lett 29, 1–16.

    Article  PubMed  CAS  Google Scholar 

  8. Durot M, Bourguignon PY, and Schachter V (2009) Genome-scale models of bacterial metabolism: reconstruction and applications. FEMS Microbiol Rev 33, 164–190.

    Article  PubMed  CAS  Google Scholar 

  9. Bolten CJ, Kiefer P, Letisse F, Portais JC, and Wittmann C (2007) Sampling for metabolome analysis of microorganisms. Anal Chem 79, 3843–3849.

    Article  PubMed  CAS  Google Scholar 

  10. Meyer H, Liebeke M, and Lalk M (2010) A protocol for the investigation of the intracellular Staphylococcus aureus metabolome. Anal Biochem 401, 250–259.

    Article  PubMed  CAS  Google Scholar 

  11. Donat S, Streker K, Schirmeister T, Rakette S, Stehle T, Liebeke M, Lalk M, and Ohlsen K (2009) Transcriptome and functional analysis of the eukaryotic-type serine/threonine kinase PknB in Staphylococcus aureus. J Bacteriol 191, 4056–4069.

    Article  PubMed  CAS  Google Scholar 

  12. Liebeke M, Meyer H, Donat S, Ohlsen K, and Lalk M (2010) A metabolomic view of Staphylococcus aureus and its serine/threonine kinase and phosphatase deletion mutants: involvement in cell wall biosynthesis. Chem Biol 17, 820–830.

    Article  PubMed  CAS  Google Scholar 

  13. Winder CL, Dunn WB, Schuler S, Broadhurst D, Jarvis R, Stephens GM, and Goodacre R (2008) Global metabolic profiling of Escherichia coli cultures: an evaluation of methods for quenching and extraction of intracellular metabolites. Anal Chem 80, 2939–2948.

    Article  PubMed  CAS  Google Scholar 

  14. Lisec J, Schauer N, Kopka J, Willmitzer L, and Fernie AR (2006) Gas chromatography mass spectrometry-based metabolite profiling in plants. Nat Protoc 1, 387–396.

    Article  PubMed  CAS  Google Scholar 

  15. Want EJ, Coen M, Masson P, Keun HC, Pearce JT, Reily MD, Robertson DG, Rohde CM, Holmes E, Lindon JC, Plumb RS, and Nicholson JK (2010) Ultra performance liquid chromatography-mass spectrometry profiling of bile acid metabolites in biofluids: application to experimental toxicology studies. Anal Chem 82, 5282–5289.

    Article  PubMed  CAS  Google Scholar 

  16. Villas-Boas SG, Mas S, Akesson M, Smedsgaard J, and Nielsen J (2005) Mass spectrometry in metabolome analysis. Mass Spectrom Rev 24, 613–646.

    Article  PubMed  CAS  Google Scholar 

  17. Cubbon S, Antonio C, Wilson J, and Thomas-Oates J (2010) Metabolomic applications of HILIC-LC-MS. Mass Spectrom Rev 29, 671–684.

    Article  PubMed  CAS  Google Scholar 

  18. Allwood JW, and Goodacre R (2009) An introduction to liquid chromatography-mass spectrometry instrumentation applied in plant metabolomic analyses. Phytochem Anal 21, 33–47.

    Article  Google Scholar 

  19. Bajad SU, Lu W, Kimball EH, Yuan J, Peterson C, and Rabinowitz JD (2006) Separation and quantitation of water soluble cellular metabolites by hydrophilic interaction chromatography-tandem mass spectrometry. J Chromatogr A 1125, 76–88.

    Article  PubMed  CAS  Google Scholar 

  20. Buescher JM, Moco S, Sauer U, and Zamboni N (2010) Ultrahigh performance liquid chromatography-tandem mass spectrometry method for fast and robust quantification of anionic and aromatic metabolites. Anal Chem 82, 4403–4412.

    Article  PubMed  CAS  Google Scholar 

  21. Roessner U, Luedemann A, Brust D, Fiehn O, Linke T, Willmitzer L, and Fernie A (2001) Metabolic profiling allows comprehensive phenotyping of genetically or environmentally modified plant systems. Plant Cell 13, 11–29.

    Article  PubMed  CAS  Google Scholar 

  22. Roessner-Tunali U, Urbanczyk-Wochniak E, Czechowski T, Kolbe A, Willmitzer L, and Fernie AR (2003) De novo amino acid biosynthesis in potato tubers is regulated by sucrose levels. Plant Physiol 133, 683–692.

    Article  PubMed  CAS  Google Scholar 

  23. Sangster T, Major H, Plumb R, Wilson AJ, and Wilson ID (2006) A pragmatic and readily implemented quality control strategy for HPLC-MS and GC-MS-based metabonomic analysis. Analyst 131, 1075–1078.

    Article  PubMed  CAS  Google Scholar 

  24. Wu L, Mashego MR, van Dam JC, Proell AM, Vinke JL., Ras C, van Winden WA, van Gulik W M, and Heijnen JJ (2005) Quantitative analysis of the microbial metabolome by isotope dilution mass spectrometry using uniformly 13C-labeled cell extracts as internal standards. Anal Biochem 336, 164–171.

    Article  PubMed  CAS  Google Scholar 

  25. Bennett BD, Yuan J, Kimball EH, and Rabinowitz JD (2008) Absolute quantitation of intracellular metabolite concentrations by an isotope ratio-based approach. Nat Protoc 3, 1299–1311.

    Article  PubMed  CAS  Google Scholar 

  26. Pluskal T, Nakamura T, Villar-Briones A, and Yanagida M Metabolic profiling of the fission yeast S. pombe: quantification of compounds under different temperatures and genetic perturbation. Mol Biosyst 6, 182–198.

    Google Scholar 

  27. Liebeke M, Brozel VS, Hecker M, and Lalk M (2009) Chemical characterization of soil extract as growth media for the ecophysiological study of bacteria. Appl Microbiol Biotechnol 83, 161–173.

    Article  PubMed  CAS  Google Scholar 

  28. Benton HP, Wong DM, Trauger SA, and Siuzdak G (2008) XCMS2: processing tandem mass spectrometry data for metabolite identification and structural characterization. Anal Chem 80, 6382–6389.

    Article  PubMed  CAS  Google Scholar 

  29. Lommen A (2009) MetAlign: interface-driven, versatile metabolomics tool for hyphenated full-scan mass spectrometry data preprocessing. Anal Chem 81, 3079–3086.

    Article  PubMed  CAS  Google Scholar 

  30. Bunk B, Kucklick M, Jonas R, Munch R, Schobert M, Jahn D, and Hiller K (2006) MetaQuant: a tool for the automatic quantification of GC/MS-based metabolome data. Bioinformatics 22, 2962–2965.

    Article  PubMed  CAS  Google Scholar 

  31. Luedemann A, Strassburg K, Erban A, and Kopka J (2008) TagFinder for the quantitative analysis of gas chromatography–mass spectrometry (GC–MS)-based metabolite profiling experiments. Bioinformatics 24, 732–737.

    Article  PubMed  CAS  Google Scholar 

  32. Trygg J, Holmes E, and Lundstedt T (2007) Chemometrics in metabonomics. J Proteome Res 6, 469–479.

    Article  PubMed  CAS  Google Scholar 

  33. Lai L, Michopoulos F, Gika H, Theodoridis G, Wilkinson RW, Odedra R, Wingate J, Bonner R, Tate S, and Wilson ID (2010) Methodological considerations in the development of HPLC-MS methods for the analysis of rodent plasma for metabonomic studies. Mol Biosyst 6, 108–120.

    Article  PubMed  CAS  Google Scholar 

  34. Burton L, Ivosev G, Tate S, Impey G, Wingate J, and Bonner R (2008) Instrumental and experimental effects in LC-MS-based metabolomics. J Chromatogr B 871, 227–235.

    Article  CAS  Google Scholar 

  35. Hummel J, Strehmel N, Selbig J, Walther D, and Kopka J (2010) Decision tree supported substructure prediction of metabolites from GC-MS profiles. Metabolomics 6, 322–333.

    Article  PubMed  CAS  Google Scholar 

  36. Smith CA, Want EJ, O’Maille G, Abagyan R, and Siuzdak G (2006) XCMS: processing mass spectrometry data for metabolite profiling using nonlinear peak alignment, matching, and identification. Anal Chem 78, 779–787.

    Article  PubMed  CAS  Google Scholar 

  37. Katajamaa M, Miettinen J, and Oresic M (2006) MZmine: toolbox for processing and visualization of mass spectrometry based molecular profile data. Bioinformatics 22, 634–636.

    Article  PubMed  CAS  Google Scholar 

  38. Sumner LW, Urbanczyk-Wochniak E, and Broeckling CD (2007) Metabolomics data analysis, visualization, and integration. Methods Mol Biol 406, 409–436.

    Article  PubMed  CAS  Google Scholar 

  39. Smith CA, O’Maille G, Want EJ, Qin C, Trauger SA, Brandon TR, Custodio DE, Abagyan R, and Siuzdak G (2005) METLIN: a metabolite mass spectral database. Ther Drug Monit 27, 747–751.

    Article  PubMed  CAS  Google Scholar 

  40. Ulrich EL, Akutsu H, Doreleijers JF, Harano Y, Ioannidis YE, Lin J, Livny M, Mading S, Maziuk D, Miller Z, Nakatani E, Schulte CF, Tolmie DE, Kent Wenger R, Yao H, and Markley JL (2008) BioMagResBank. Nucleic Acids Res 36, D402–408.

    Article  PubMed  CAS  Google Scholar 

  41. Wishart, DS, Knox C, Guo AC, Eisner R, Young N, Gautam B, Hau DD, Psychogios N, Dong E, Bouatra S, Mandal R, et al. (2009) HMDB: a knowledgebase for the human metabolome. Nucleic Acids Res 37, D603–610.

    Article  PubMed  CAS  Google Scholar 

  42. Atkinson DE (1968) The energy charge of the adenylate pool as a regulatory parameter. Interaction with feedback modifiers. Biochemistry-Us 7, 4030–4034.

    CAS  Google Scholar 

  43. Steuer R, Morgenthal K, Weckwerth W, and Selbig J (2007) A gentle guide to the analysis of metabolomic data. Methods Mol Biol 358, 105–126.

    Article  PubMed  CAS  Google Scholar 

  44. Gehlenborg N, O’Donoghue SI, Baliga NS, Goesmann A, Hibbs MA, Kitano H, Kohlbacher O, Neuweger H, Schneider R, Tenenbaum D, and Gavin AC (2010) Visualization of omics data for systems biology. Nat Methods 7, S56–68.

    Article  PubMed  CAS  Google Scholar 

  45. Scholz M, and Selbig J (2007) Visualization and analysis of molecular data. Methods Mol Biol 358, 87–104.

    Article  PubMed  CAS  Google Scholar 

  46. Bligh EG, and Dyer WJ (1959) A rapid method of total lipid extraction and purification. Can J Biochem Physiol 37, 911–917.

    Article  PubMed  CAS  Google Scholar 

  47. Kouremenos KA, Harynuk JJ, Winniford WL, Morrison PD, and Marriott PJ (2010) One-pot microwave derivatization of target compounds relevant to metabolomics with comprehensive two-dimensional gas chromatography. J Chromatogr B 878, 1761–1770.

    Article  CAS  Google Scholar 

  48. Liebeke M, Wunder A, and Lalk M (2010) A rapid microwave-assisted derivatization of bacterial metabolome samples for GC/MS analysis. Anal Biochem 401, 312–314.

    Article  PubMed  CAS  Google Scholar 

  49. Coulier L, Bas R, Jespersen S, Verheij E, van der Werf MJ, and Hankemeier T (2006) Simultaneous quantitative analysis of metabolites using ion-pair liquid chromatography-electrospray ionization mass spectrometry. Anal Chem 78, 6573–6582.

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Biomolecular Medicine, Department of Surgery and Cancer, Faculty of Medicine, Imperial College London, London, SW7 2AZ, UK

    Manuel Liebeke

  2. Institute of Pharmacy, Ernst-Moritz-Arndt-University Greifswald, Friedrich-Ludwig-Jahn-Str. 17, Greifswald, 17487, Germany

    Kirsten Dörries

  3. Institute of Pharmacy, Interfaculty Institute for Genetics and Functional Genomics, University of Greifswald, F.-L.-Jahnstr. 15, Greifswald, D-17487, Germany

    Hanna Meyer & Michael Lalk

Authors
  1. Manuel Liebeke
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Kirsten Dörries
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Hanna Meyer
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Michael Lalk
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Manuel Liebeke .

Editor information

Editors and Affiliations

  1. School of Medicine, Witten/Herdecke University, Group Stockumer Str. 10 50, Witten, 58448, Germany

    Michael Kaufmann

  2. School of Medicine, Private Universität Witten/Herdecke, Witten/Herdecke University 50, Witten, 58448, Germany

    Claudia Klinger

Rights and permissions

Reprints and permissions

Copyright information

© 2012 Springer Science+Business Media, LLC

About this protocol

Cite this protocol

Liebeke, M., Dörries, K., Meyer, H., Lalk, M. (2012). Metabolome Analysis of Gram-Positive Bacteria such as Staphylococcus aureus by GC-MS and LC-MS. In: Kaufmann, M., Klinger, C. (eds) Functional Genomics. Methods in Molecular Biology, vol 815. Springer, New York, NY. https://doi.org/10.1007/978-1-61779-424-7_28

Download citation

  • DOI: https://doi.org/10.1007/978-1-61779-424-7_28

  • Published:

  • Publisher Name: Springer, New York, NY

  • Print ISBN: 978-1-61779-423-0

  • Online ISBN: 978-1-61779-424-7

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation