Mass Spectrometry-Based Microbial Metabolomics

  • Edward E. K. Baidoo
  • Peter I. Benke
  • Jay D. KeaslingEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 881)


Through the characterization of metabolic pathways, metabolomics is able to illuminate the activities of a cell at the functional level. However, the metabolome, which is comprised of hundreds of chemically diverse metabolites, is rather difficult to monitor. Mass spectrometry (MS)-based profiling methods are increasingly being utilized for this purpose. To this end, MS is often coupled to the separation techniques gas chromatography (GC), liquid chromatography (LC), and capillary electrophoresis (CE). The mass-based selectivity that the MS provides, together with the chromatographic or electrophoretic separation of analytes, creates hyphenated techniques that are ideally suited to the measurement of large numbers of metabolites from microbial extracts. In this chapter, we describe GC-MS, LC-MS, and CE-MS methods that are applicable to microbial metabolomics experiments.

Key words

Metabolomics Mass spectrometry Quenching Extraction GC-MS LC-MS CE-MS 



This work conducted by ENIGMA—Ecosystems and Networks Integrated with Genes and Molecular Assemblies—was supported by the Office of Science, Office of Biological and Environmental Research, of the U.S. Department of Energy under Contract No. DE-AC02-05CH11231.

This work was also part of the DOE Joint BioEnergy Institute ( supported by the Office of Science, Office of Biological and Environmental Research, of the U.S. Department of Energy through Contract No. DE-AC02-05CH11231 between Lawrence Berkeley National Laboratory and the U.S. Department of Energy.


  1. 1.
    Villas-Bôas SG, Roessner U, Hansen MAE, Smedsgaard J, Nielsen J (2007) Metabolome analysis: an introduction. Wiley, HobokenGoogle Scholar
  2. 2.
    Warwick BD, Ellis DI (2005) Metabolomics: current analytical platforms and methodologies. Trends Anal Chem 24:285–294CrossRefGoogle Scholar
  3. 3.
    Mashego MR, Rumbold K, De Mey M, Vandamme E, Soetaert W, Heijnen JJ (2007) Microbial metabolomics: past, present and future methodologies. Biotechnol Lett 29:1–16PubMedCrossRefGoogle Scholar
  4. 4.
    Bender DA (2005) Perspective. The promise of metabolomics. J Sci Food Agric 85:7–9CrossRefGoogle Scholar
  5. 5.
    Griffin JL (2004) Metabolic profiles to define the genome: can we hear the phenotypes? Philos Trans R Soc Lond B Biol Sci 359:857–871PubMedCrossRefGoogle Scholar
  6. 6.
    Borodina I, Nielsen J (2005) From genomes to in silico cells via metabolic networks. Curr Opin Biotechnol 16:350–355PubMedCrossRefGoogle Scholar
  7. 7.
    Schmidt C (2004) Metabolomics takes its place as latest up-and-coming “omic” science. J Natl Cancer Inst 96:732–734PubMedCrossRefGoogle Scholar
  8. 8.
    Brock TD, Madigan MT, Martinko JM, Parker J (1994) Brock biology of microorganisms. Prentice Hall, Englewood CliffsGoogle Scholar
  9. 9.
    Skoog DA, Holler FJ, Nieman TA (2006) Principles of instrumental analysis, 6th edn. Brooks Cole, Pacific GroveGoogle Scholar
  10. 10.
    Harris DC (2003) Quantitative chemical analysis, 6th edn. W.H. Freeman and Company, New YorkGoogle Scholar
  11. 11.
    Fjeldsted J (2003) Time-of-flight mass spectrometry. Technical overview. Accessed 25 June 2007
  12. 12.
    de Hoffman E, Stroobant V (2002) Mass spectrometry. Principles and applications, 2nd edn. Wiley, New YorkGoogle Scholar
  13. 13.
    Stewart II (1999) Electrospray mass spectrometry: a tool for elemental speciation. Spectrochim Acta B 54:1649–1695CrossRefGoogle Scholar
  14. 14.
    Smyth WF (1999) The use of electrospray mass spectrometry in the detection and determination of molecules of biological significance. Trends Anal Chem 18:335–346CrossRefGoogle Scholar
  15. 15.
    Smith JN, Flagan RC, Beauchamp JL (2002) Droplet evaporation and discharge dynamics in electrospray ionization. J Phys Chem A 106:9957–9967CrossRefGoogle Scholar
  16. 16.
    Wilm MS, Mann M (1994) Electrospray and Taylor-Cone theory, Dole’s beam of macromolecules at last? Int J Mass Spectrom Ion Process 136:167–180CrossRefGoogle Scholar
  17. 17.
    Fenn JB (1993) Ion formation from charged droplets: roles of geometry, energy, and time. J Am Soc Mass Spectrom 4:524–535CrossRefGoogle Scholar
  18. 18.
    Garrod JW, Jacob P III (1999) Analytical determination of nicotine and related compounds and their metabolites. Elsevier Science B.V., Amsterdam, The NetherlandsGoogle Scholar
  19. 19.
    Hübschmann HJ (2009) Handbook of GC/MS. Wiley, WeinheimGoogle Scholar
  20. 20.
    McLafferty FW (1993) Interpretation of mass spectra. University Science Books, BerkeleyGoogle Scholar
  21. 21.
    Kind T, Wohlgemuth G, Lee DY, Lu Y, Palazoglu M, Shahbaz S, Fiehn O (2009) FiehnLib: mass spectral and retention index libraries for metabolomics based on quadrupole and time-of-flight gas chromatography/mass spectrometry. Anal Chem 81:10038–10048PubMedCrossRefGoogle Scholar
  22. 22.
    Kopka J, Schauer N, Krueger S, Birkemeyer C, Usadel B, Bergmuller E, Dormann P, Weckwerth W, Gibon Y, Stitt M, Willmitzer L, Fernie AR, Steinhauser D (2005) GMD@CSB.DB: the Golm metabolome database. Bioinformatics 21:1635–1638PubMedCrossRefGoogle Scholar
  23. 23.
    Willoughby R, Sheehan E, Mitrovich S (1998) A global view of LC/MS: how to solve your most challenging analytical problems, 1st edn. Global View Publishing, PittsburghGoogle Scholar
  24. 24.
    Jhonstone RAW, Rose ME (1996) Mass spectrometry for chemists and biochemists, 2nd edn. Cambridge University Press, CambridgeCrossRefGoogle Scholar
  25. 25.
    Smith RM, Busch KL (1999) Understanding mass spectra—a basic approach. Wiley, New YorkGoogle Scholar
  26. 26.
    Soga T, Ohashi Y, Ueno Y, Naraoka H, Matsuda K, Tomita M, Nishioka T (2003) Quantitative metabolome analysis using capillary electrophoresis mass spectrometry. J Proteome Res 2:488–494PubMedCrossRefGoogle Scholar
  27. 27.
    Bajad SU, Lu W, Kimball EH, Yuan J, Peterson C, Rabinowitz JD (2006) Separation and quantitation of water soluble cellular metabolites by hydrophilic interaction chromatography-tandem mass spectrometry. J Chromatogr A 1125:76–88PubMedCrossRefGoogle Scholar
  28. 28.
    Stöckigt J, Sheludko Y, Unger M, Gerasimenko I, Warzecha H, Stöckigt D (2002) High-performance liquid chromatographic, capillary electrophoretic and capillary electrophoretic-electrospray ionisation mass spectrometric analysis of selected alkaloid groups. J Chromatogr A 967:85–113PubMedCrossRefGoogle Scholar
  29. 29.
    Jonscher KR, Yates JR III (1997) The quadrupole ion trap mass spectrometer—a small solution to a big challenge. Anal Biochem 244:1–15PubMedCrossRefGoogle Scholar
  30. 30.
    Weickhardt C, Moritz F, Grotemeyer J (1996) Time-of-flight mass spectrometry: state-of the-art in chemical analysis and molecular science. Mass Spectrom Rev 15:139–162CrossRefGoogle Scholar
  31. 31.
    McIntire D (2004) Effect of resolution and mass accuracy on empirical formula confirmation and identification of unknowns. Technical overview. Accessed 20 April 2005
  32. 32.
    Giddings JC (2002) Dynamics of chromatography: principles and theory. CRC Press, DanversGoogle Scholar
  33. 33.
    Baker DR (1995) Capillary electrophoresis. Wiley, New YorkGoogle Scholar
  34. 34.
    Fiehn O (2008) Extending the breadth of metabolite profiling by gas chromatography coupled to mass spectrometry. Anal Chem 27:261–269Google Scholar
  35. 35.
    Koek MM, Muilwijk B, van Stee LLP, Hankemeier T (2008) Higher mass loadability in comprehensive two-dimensional gas chromatography-mass spectrometry for improved analytical performance in metabolomics analysis. J Chromatogr A 1186:420–429PubMedCrossRefGoogle Scholar
  36. 36.
    Blau K, Halket JM (eds) (1993) Handbook of derivatives for chromatography. Wiley, New YorkGoogle Scholar
  37. 37.
    Kanani HH, Klappa MI (2007) Data correction strategy for metabolomics analysis using gas chromatography-mass spectrometry. Metab Eng 9:39–51PubMedCrossRefGoogle Scholar
  38. 38.
    Little JL (1999) Artifacts in trimethylsilyl derivatization reactions and ways to avoid them. J Chromatogr A 844:1–22PubMedCrossRefGoogle Scholar
  39. 39.
    Kanani H, Chrysanthopoulos PK, Klapa MI (2008) Standardizing GC-MS metabolomics. J Chromatogr B 871:191–201CrossRefGoogle Scholar
  40. 40.
    Halket JM, Zaikin VG (2003) Derivatization in mass spectrometry-1. Silylation. Eur J Mass Spectrom 9:1–21CrossRefGoogle Scholar
  41. 41.
    Liebeke M, Wunder A, Lalk M (2010) A rapid microwave-assisted derivatization of bacterial metabolome samples for gas chromatography/mass spectrometry analysis. Anal Biochem 401:312–314PubMedCrossRefGoogle Scholar
  42. 42.
    Phillips JB, Beens J (1999) Comprehensive two-dimensional gas chromatography: a hyphenated method with strong coupling between the two dimensions. J Chromatogr A 856:331–347PubMedCrossRefGoogle Scholar
  43. 43.
    Mondello L, Tranchida PQ, Dugo P, Dugo G (2008) Comprehensive two-dimensional gas chromatography-mass spectrometry: a review. Mass Spectrom Rev 27:101–124PubMedCrossRefGoogle Scholar
  44. 44.
    Mohler RE, Dombek KM, Hoggard JC, Young ET, Synovec RE (2006) Comprehensive two-dimensional gas chromatography time-of-flight mass spectrometry analysis of metabolites in fermenting and respiring yeast cells. Anal Chem 78:2700–2709PubMedCrossRefGoogle Scholar
  45. 45.
    Mohler RE, Dombek KM, Hoggard JC, Pierce KM, Young ET, Synovec RE (2007) Comprehensive analysis of yeast metabolite GC × GC-TOFMS data: combining discovery-mode and deconvolution chemometric software. Analyst 132:756–767PubMedCrossRefGoogle Scholar
  46. 46.
    Lu H, Liang Y, Dunn WB, Shen H, Kell DB (2008) Comparative evaluation of software for deconvolution of metabolomics data based on GC-TOF-MS. Trends Anal Chem 27:215–227CrossRefGoogle Scholar
  47. 47.
    Scalbert A, Brennan L, Fiehn O, Hankemeier T, Kristal BS, van Ommen B, Pujos-Guillot E, Verheij E, Wishart D, Wopereis S (2009) Mass-spectrometry-based metabolomics: limitations and recommendations for future progress with particular focus on nutrition research. Metabolomics 5:435–458PubMedCrossRefGoogle Scholar
  48. 48.
    Mac-Mod Analytical Inc. Accessed 21 June 2010
  49. 49.
    Snyder LR, Kirkland JJ, Dolan JW (2010) Introduction to modern liquid chromatography. Wiley, HobokenGoogle Scholar
  50. 50.
    Guillarme D, Schappler J, Rudaz S, Veuthey J-L (2010) Coupling ultra-high-pressure liquid chromatography with mass spectrometry. Trends Anal Chem 29:15–27CrossRefGoogle Scholar
  51. 51.
    Superficially porous HPLC columns and column overload, Technical Overview (2010) 5990-6001EN.
  52. 52.
    Cunliffe JM, Maloney TD (2007) Fused-core particle technology as an alternative to sub-2-μm particles to achieve high separation efficiency with low backpressure. J Sep Sci 30:3104–3109PubMedCrossRefGoogle Scholar
  53. 53.
    Aurand CR, Bell DS, Lamb T, Bell-Pedersen D (2010) Metabolomic profiling of Neurospora crassa fungi using HILIC and reversed-phase LC-MS. Reporter US 28(3):14–15. Google Scholar
  54. 54.
    Alpert AJ (2008) Electrostatic repulsion hydrophilic interaction chromatography for isocratic separation of charged solutes and selective isolation of phosphopeptides. Anal Chem 80:62–76PubMedCrossRefGoogle Scholar
  55. 55.
    Luo B, Groenke K, Takors R, Wandrey C, Oldiges M (2007) Simultaneous determination of multiple intracellular metabolites in glycolysis, pentose phosphate pathway and tricarboxylic acid cycle by liquid chromatography-mass spectrometry. J Chromatogr A 1147:153–164PubMedCrossRefGoogle Scholar
  56. 56.
    Bennett BD, Kimball EH, Gao M, Osterhout R, Van Dien SJ, Rabinowitz JD (2009) Absolute metabolite concentrations and implied enzyme active site occupancy in Escherichia coli. Nat Chem Biol 5:593–599PubMedCrossRefGoogle Scholar
  57. 57.
    Lu W, Kimball E, Rabinowitz JD (2006) A high-performance liquid chromatography-tandem mass spectrometry method for quantitation of nitrogen-containing intracellular metabolites. J Am Soc Mass Spectrom 17:37–50PubMedCrossRefGoogle Scholar
  58. 58.
    Buescher JM, Moco S, Sauer U, Zamboni N (2010) Ultrahigh performance liquid chromatography—tandem mass spectrometry method for fast and robust quantification of anionic and aromatic metabolites. Anal Chem 82:4403–4412PubMedCrossRefGoogle Scholar
  59. 59.
    Ding J, Vouros P (1999) Recent developments in interfaces and applications. Anal Chem 71:378A–385APubMedCrossRefGoogle Scholar
  60. 60.
    Chang YZ, Her GR (2000) Sheathless capillary electrophoresis/electrospray mass spectrometry using a carbon-coated fused-silica capillary. Anal Chem 72:626–630PubMedCrossRefGoogle Scholar
  61. 61.
    Banks JF (1997) Recent advances in capillary electrophoresis/electrospray/mass spectrometry. Electrophoresis 18:2255–2266PubMedCrossRefGoogle Scholar
  62. 62.
    Tomer KB (2001) Separations combined with mass spectrometry. Chem Rev 101:297–328PubMedCrossRefGoogle Scholar
  63. 63.
    Lausecker B, Hopfgartner G, Hesse M (1998) Capillary electrophoresis-mass spectrometry coupling versus micro-high-performance liquid chromatography-mass spectrometry coupling: a case study. J Chromatogr B 718:1–13CrossRefGoogle Scholar
  64. 64.
    Baidoo EK, Benke PI, Neusüss C, Pelzing M, Kruppa G, Leary JA, Keasling JD (2008) Capillary electrophoresis-Fourier transform ion cyclotron resonance mass spectrometry for the identification of cationic metabolites via a pH-mediated stacking-transient isotachophoretic method. Anal Chem 80:3112–3122. Copyright © 2008, with permission from American Chemical SocietyGoogle Scholar
  65. 65.
    Harada K, Fukusaki E, Kobayashi A (2006) Pressure-assisted capillary electrophoresis mass spectrometry using combination of polarity reversion and electroosmotic flow for metabolomics anion analysis. J Biosci Bioeng 101:403–409PubMedCrossRefGoogle Scholar
  66. 66.
    Ohashi Y, Hirayama A, Ishikawa T, Nakamura S, Shimizu K, Ueno Y, Tomita M, Soga T (2007) Depiction of metabolome changes in histidine-starved Escherichia coli by CE-TOFMS. Mol Biosyst 4:135–147PubMedCrossRefGoogle Scholar
  67. 67.
    Rabinowitz JD, Kimball E (2007) Acidic acetonitrile for cellular metabolome extraction from Escherichia coli. Anal Chem 79:6167–6173PubMedCrossRefGoogle Scholar
  68. 68.
    Soga T, Ueno Y, Naraoka H, Matsuda K, Tomita M, Nishioka T (2002) Pressure-assisted capillary electrophoresis electrospray ionization mass spectrometry for analysis of multivalent anions. Anal Chem 74:6224–6229PubMedCrossRefGoogle Scholar
  69. 69.
    Voyksner RD, Lee H (1999) Improvements in LC/electrospray ion trap mass spectrometry performance using an off-axis nebulizer. Anal Chem 71:1441–1447PubMedCrossRefGoogle Scholar
  70. 70.
    Soga T, Ueno Y, Naraoka H, Ohasi Y, Tomita M, Nishioka T (2002) Simultaneous determination of anionic intermediates for Bacillus subtilis metabolic pathways by capillary electrophoresis electrospray ionization mass spectrometry. Anal Chem 74:2233–2239PubMedCrossRefGoogle Scholar
  71. 71.
    Soga T, Ishikawa T, Igarashi S, Sugawara K, Kakazu Y, Tomita M (2007) Analysis of nucleotides by pressure-assisted capillary electrophoresis-mass spectrometry using silanol mask technique. J Chromatogr A 1159:125–133PubMedCrossRefGoogle Scholar
  72. 72.
    Steuer R, Morgenthal K, Weckwerth W, Selbig J (2006) A gentle guide to the analysis of metabolomics data. In: Weckwerth W (ed) Metabolomics: methods and protocols, vol 358, Methods in molecular biology. Springer, New YorkGoogle Scholar
  73. 73.
    Burgi DS, Chien RL (1991) Optimization in sample stacking for high-performance capillary electrophoresis. Anal Chem 63:2042–2047CrossRefGoogle Scholar
  74. 74.
    Van Berkel GJ (1997) The electrolytic nature of electrospray. In: Cole RB (ed) Electrospray ionization mass spectrometry: fundamentals, instrumentation, and applications. Wiley, New YorkGoogle Scholar
  75. 75.
    Soga T, Igarashi K, Ito C, Mizobuchi K, Zimmermann H-P, Tomita M (2009) Metabolomic profiling of anionic metabolites by capillary electrophoresis mass spectrometry. Anal Chem 81:6165–6174PubMedCrossRefGoogle Scholar
  76. 76.
    Brauer MJ, Yuan J, Bennett BD, Lu W, Kimball E, Botstein D, Rabinowitz JD (2006) Conservation of the metabolomic response to starvation across two divergent microbes. Proc Natl Acad Sci U S A 103:19302–19307PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Edward E. K. Baidoo
    • 1
    • 2
  • Peter I. Benke
    • 1
    • 2
  • Jay D. Keasling
    • 3
    • 4
    • 1
    • 2
    Email author
  1. 1.Physical Biosciences DivisionLawrence Berkeley National LaboratoryBerkeleyUSA
  2. 2.Joint BioEnergy InstituteEmeryvilleUSA
  3. 3.Department of Chemical EngineeringUniversity of CaliforniaBerkeleyUSA
  4. 4.Department of BioengineeringUniversity of CaliforniaBerkeleyUSA

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