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Analysis of carbon and nitrogen co-metabolism in yeast by ultrahigh-resolution mass spectrometry applying 13C- and 15N-labeled substrates simultaneously

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Abstract

Alternative metabolic pathways inside a cell can be deduced using stable isotopically labeled substrates. One prerequisite is accurate measurement of the labeling pattern of targeted metabolites. Experiments are generally limited to the use of single-element isotopes, mainly 13C. Here, we demonstrate the application of direct infusion nanospray, ultrahigh-resolution Fourier transform ion cyclotron resonance-mass spectrometry (FTICR-MS) for metabolic studies using differently labeled elemental isotopes simultaneously—i.e., 13C and 15N—in amino acids of a total protein hydrolysate. The optimized strategy for the analysis of metabolism by a hybrid linear ion trap-FTICR-MS comprises the collection of multiple adjacent selected ion monitoring scans. By limiting both the width of the mass range and the number of ions entering the ICR cell with automated gain control, sensitive measurements of isotopologue distribution were possible without compromising mass accuracy and isotope intensity mapping. The required mass-resolving power of more than 60,000 is only achievable on a routine basis by FTICR and Orbitrap mass spectrometers. Evaluation of the method was carried out by comparison of the experimental data to the natural isotope abundances of selected amino acids and by comparison to GC/MS results obtained from a labeling experiment with 13C-labeled glucose. The developed method was used to shed light on the complexity of the yeast Saccharomyces cerevisiae carbon–nitrogen co-metabolism by administering both 13C-labeled glucose and 15N-labeled alanine. The results indicate that not only glutamate but also alanine acts as an amino donor during alanine and valine synthesis. Metabolic studies using FTICR-MS can exploit new possibilities by the use of multiple-labeled elemental isotopes.

Analysis of carbon and nitrogen co-metabolism in yeast by nanospray-FTICR-MS determination of 13C and 15N incorporation into proteinogenic amino acids

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References

  1. Sauer U (2006) Metabolic networks in motion: 13C-based flux analysis. Mol Syst Biol 2:62

    Article  Google Scholar 

  2. Szyperski T (1998) C-13-NMR, MS and metabolic flux balancing in biotechnology research. Q Rev Biophys 31:41–106

    Article  CAS  Google Scholar 

  3. Wiechert W, de Graaf AA (1996) In vivo stationary flux analysis by 13C labeling experiments. Adv Biochem Eng Biotechnol 54:109–154

    CAS  Google Scholar 

  4. Tang YJ, Martin HG, Myers S, Rodriguez S, Baidoo EE, Keasling JD (2009) Advances in analysis of microbial metabolic fluxes via 13C isotopic labeling. Mass Spectrom Rev 28:362–375

    Article  CAS  Google Scholar 

  5. Mashego MR, Wu L, Van Dam JC, Ras C, Vinke JL, Van Winden WA, Van Gulik WM, Heijnen JJ (2004) MIRACLE: mass isotopomer ratio analysis of U-C-13-labeled extracts. A new method for accurate quantification of changes in concentrations of intracellular metabolites. Biotechnol Bioeng 85:620–628

    Article  CAS  Google Scholar 

  6. Birkemeyer C, Luedemann A, Wagner C, Erban A, Kopka J (2005) Metabolome analysis: the potential of in vivo labeling with stable isotopes for metabolite profiling. Trends Biotechnol 23:28–33

    Article  CAS  Google Scholar 

  7. Wittmann C, Heinzle E (1999) Mass spectrometry for metabolic flux analysis. Biotechnol Bioeng 62:739–750

    Article  CAS  Google Scholar 

  8. Wittmann C (2007) Fluxome analysis using GC-MS. Microb Cell Fact 6:6

    Article  Google Scholar 

  9. Wiechert W (2001) C-13 metabolic flux analysis. Metab Eng 3:195–206

    Article  CAS  Google Scholar 

  10. Malloy CR, Sherry AD, Jeffrey FMH (1988) Evaluation of carbon flux and substrate selection through alternate pathways involving the citric-acid cycle of the heart by C-13 NMR-spectroscopy. J Biol Chem 263:6964–6971

    CAS  Google Scholar 

  11. Zamboni N, Fischer E, Sauer U (2005) FiatFlux—a software for metabolic flux analysis from 13C-glucose experiments. BMC Bioinforma 6:209

    Article  Google Scholar 

  12. Wiechert W, Möllney M, Petersen S, de Graaf AA (2001) A universal framework for 13C metabolic flux analysis. Metab Eng 3:265–283

    Article  CAS  Google Scholar 

  13. Noh K, Gronke K, Luo B, Takors R, Oldiges M, Wiechert W (2007) Metabolic flux analysis at ultra short time scale: isotopically non-stationary C-13 labeling experiments. J Biotechnol 129:249–267

    Article  Google Scholar 

  14. Schaub J, Mauch A, Reuss M (2008) Metabolic flux analysis in Escherichia coli by integrating isotopic dynamic and isotopic stationary C-13 labeling data. Biotechnol Bioeng 99:1170–1185

    Article  CAS  Google Scholar 

  15. van Winden WA, van Dam JC, Ras C, Kleijn RJ, Vinke JL, van Gulik WM, Heijnen JJ (2005) Metabolic-flux analysis of Saccharomyces cerevisiae CEN.PK113-7D based on mass isotopomer measurements of C-13-labeled primary metabolites. FEMS Yeast Res 5:559–568

    Article  Google Scholar 

  16. Toya Y, Ishii N, Hirasawa T, Naba M, Hirai K, Sugawara K, Igarashi S, Shimizu K, Tomita M, Soga T (2007) Direct measurement of isotopomer of intracellular metabolites using capillary electrophoresis time-of-flight mass spectrometry for efficient metabolic flux analysis. J Chromatogr A 1159:134–141

    Article  CAS  Google Scholar 

  17. Yuan J, Fowler WU, Kimball E, Lu WY, Rabinowitz JD (2006) Kinetic flux profiling of nitrogen assimilation in Escherichia coli. Nat Chem Biol 2:529–530

    Article  CAS  Google Scholar 

  18. Engelsberger WR, Erban A, Kopka J, Schulze WX (2006) Metabolic labeling of plant cell cultures with K(15)NO3 as a tool for quantitative analysis of proteins and metabolites. Plant Methods 2:14

    Article  Google Scholar 

  19. Harada K, Fukusaki E, Bamba T, Sato F, Kobayashi A (2006) In vivo 15N-enrichment of metabolites in suspension cultured cells and its application to metabolomics. Biotechnol Prog 22:1003–1011

    Article  CAS  Google Scholar 

  20. Schwender J, Shachar-Hill Y, Ohlrogge JB (2006) Mitochondrial metabolism in developing embryos of Brassica napus. J Biol Chem 281:34040–34047

    Article  CAS  Google Scholar 

  21. Godin JP, Mermoud AF, Remond D, Faure M, Breuille D, Williamson G, Pere-Trepat E, Ramadan Z, Fay LB, Kochhar S (2009) Simultaneous measurement of 13C- and 15N-isotopic enrichments of threonine by mass spectrometry. Rapid Commun Mass Spectrom 23:1109–1115

    Article  CAS  Google Scholar 

  22. Popa R, Weber PK, Pett-Ridge J, Finzi JA, Fallon SJ, Hutcheon ID, Nealson KH, Capone DG (2007) Carbon and nitrogen fixation and metabolite exchange in and between individual cells of Anabaena oscillarioides. ISME J 1:354–360

    CAS  Google Scholar 

  23. Pinto DM, Boyd RK, Volmer DA (2002) Ultra-high resolution for mass spectrometric analysis of complex and low-abundance mixtures—the emergence of FTICR-MS as an essential analytical tool. Anal Bioanal Chem 373:378–389

    Article  CAS  Google Scholar 

  24. Bergquist J (2003) FTICR mass spectrometry in proteomics. Curr Opin Mol Ther 5:310–314

    CAS  Google Scholar 

  25. Dettmer K, Aronov PA, Hammock BD (2007) Mass spectrometry-based metabolomics. Mass Spectrom Rev 26:51–78

    Article  CAS  Google Scholar 

  26. Brown SC, Kruppa G, Dasseux JL (2005) Metabolomics applications of FT-ICR mass spectrometry. Mass Spectrom Rev 24:223–231

    Article  CAS  Google Scholar 

  27. Pingitore F, Tang Y, Kruppa GH, Keasling JD (2007) Analysis of amino acid isotopomers using FT-ICR MS. Anal Chem 79:2483–2490

    Article  CAS  Google Scholar 

  28. Tang YJ, Pingitore F, Mukhopadhyay A, Phan R, Hazen TC, Keasling JD (2007) Pathway confirmation and flux analysis of central metabolic pathways in Desulfovibrio vulgaris hildenborough using gas chromatography–mass spectrometry and Fourier transform-ion cyclotron resonance mass spectrometry. J Bacteriol 189:940–949

    Article  CAS  Google Scholar 

  29. Nakamura Y, Kimura A, Saga H, Oikawa A, Shinbo Y, Kai K, Sakurai N, Suzuki H, Kitayama M, Shibata D, Kanaya S, Ohta D (2007) Differential metabolomics unraveling light/dark regulation of metabolic activities in Arabidopsis cell culture. Planta 227:57–66

    Article  CAS  Google Scholar 

  30. Southam AD, Payne TG, Cooper HJ, Arvanitis TN, Viant MR (2007) Dynamic range and mass accuracy of wide-scan direct infusion-nanoelectrospray fourier transform ion cyclotron resonance mass spectrometry-based metabolomics increased by the spectral stitching method. Anal Chem 79:4595–4602

    Article  CAS  Google Scholar 

  31. Oikawa A, Nakamura Y, Ogura T, Kimura A, Suzuki H, Sakurai N, Shinbo Y, Shibata D, Kanaya S, Ohta D (2006) Clarification of pathway-specific inhibition by Fourier transform ion cyclotron resonance/mass spectrometry-based metabolic phenotyping studies. Plant Physiol 142:398–413

    Article  CAS  Google Scholar 

  32. Mungur R, Glass AD, Goodenow DB, Lightfoot DA (2005) Metabolite fingerprinting in transgenic Nicotiana tabacum altered by the Escherichia coli glutamate dehydrogenase gene. J Biomed Biotechnol 2005:198–214

    Article  CAS  Google Scholar 

  33. Zamboni N, Fendt S-M, Rühl M, Sauer U (2009) 13C-based metabolic flux analysis. Nat Protoc 4:878–892

    Article  CAS  Google Scholar 

  34. Quek LE, Dietmair S, Krömer JO, Nielsen LK (2010) Metabolic flux analysis in mammalian cell culture. Metab Eng 12:161–171

    Article  CAS  Google Scholar 

  35. Verduyn C, Postma E, Scheffers WA, Van Dijken JP (1992) Effect of benzoic acid on metabolic fluxes in yeasts: a continuous-culture study on the regulation of respiration and alcoholic fermentation. Yeast 8:501–517

    Article  CAS  Google Scholar 

  36. Raghevendran V, Gombert AK, Christensen B, Kötter P, Nielsen J (2004) Phenotypic characterization of glucose repression mutants of Saccharomyces cerevisiae using experiments with 13C-labelled glucose. Yeast 21:769–779

    Article  CAS  Google Scholar 

  37. Blank LM, Lehmbeck F, Sauer U (2005) Metabolic flux and network analysis in 14 hemiascomycetous yeasts. FEMS Yeast Res 5:545–558

    Article  CAS  Google Scholar 

  38. Heyland J, Fu J, Blank LM (2009) Correlation between TCA cycle flux and glucose uptake rate during respiro-fermentative growth of Saccharomyces cerevisiae. Microbiology 155:3827–3837

    Article  CAS  Google Scholar 

  39. Fischer E, Sauer U (2003) Metabolic flux profiling of Escherichia coli mutants in central carbon metabolism using GC-MS. Eur J Biochem 270:880–891

    Article  CAS  Google Scholar 

  40. Gancedo JM (1998) Yeast carbon catabolite repression. Microbiol Mol Biol Rev 62:334–361

    CAS  Google Scholar 

  41. Dauner M, Sauer U (2000) GC-MS analysis of amino acids rapidly provides rich information for isotopomer balancing. Biotechnol Prog 16:642–649

    Article  CAS  Google Scholar 

  42. Usaite R, Patil KR, Grotkjaer T, Nielsen J, Regenberg B (2006) Global transcriptional and physiological responses of Saccharomyces cerevisiae to ammonium, l-alanine, or l-glutamine limitation. Appl Environ Microbiol 72:6194–6203

    Article  CAS  Google Scholar 

  43. Karas M, Bahr U, Dulcks T (2000) Nano-electrospray ionization mass spectrometry: addressing analytical problems beyond routine. Fresenius J Anal Chem 366:669–676

    Article  CAS  Google Scholar 

  44. Wilm M, Mann M (1996) Analytical properties of the nanoelectrospray ion source. Anal Chem 68:1–8

    Article  CAS  Google Scholar 

  45. Fligge TA, Bruns K, Przybylski M (1998) Analytical development of electrospray and nanoelectrospray mass spectrometry in combination with liquid chromatography for the characterization of proteins. J Chromatogr B Biomed Sci Appl 706:91–100

    Article  CAS  Google Scholar 

  46. Marshall AG, Hendrickson CL (2008) High-resolution mass spectrometers. Annu Rev Anal Chem 1:579–599

    Article  CAS  Google Scholar 

  47. Colón M, Hernández F, López K, Quezada H, González J, López G, Aranda C, González A (2011) Saccharomyces cerevisiae Bat1 and Bat2 aminotransferases have functionally diverged from the ancestral-like Kluyveromyces lactis orthologous enzyme. PLoS One 18:e16099

    Article  Google Scholar 

  48. Van Pelt CK, Zhang S, Henion JD (2002) Characterization of a fully automated nanoelectrospray system with mass spectrometric detection for proteomic analyses. Biomol Technol 13:72–84

    Google Scholar 

  49. Cherry JM, Adler C, Ball C, Chervitz SA, Dwight SS, Hester ET, Jia Y, Juvik G, Roe T, Schroeder M, Weng S, Botstein D (1998) SGD: Saccharomyces Genome Database. Nucleic Acids Res 26:73–79

    Article  CAS  Google Scholar 

  50. García-Campusano F, Anaya VH, Robledo-Arratia L, Quezada H, Hernández H, Riego L, González A (2009) ALT1-encoded alanine aminotransferase plays a central role in the metabolism of alanine in Saccharomyces cerevisiae. Can J Microbiol 55:368–374

    Article  Google Scholar 

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Acknowledgments

Financial support by the “Ministerium für Innovation, Wissenschaft, Forschung und Technologie des Landes Nordrhein-Westfalen” and by the “Bundesministerium für Bildung und Forschung” is gratefully acknowledged. The authors thank Ms. Helma Geltenpoth for the excellent technical support.

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Author notes

  1. Lars M. Blank

    Present address: Institute of Applied Microbiology—iAMB, Aachen Biology and Biotechnology—ABBt, RWTH Aachen University, 52062, Aachen, Germany

  2. Rahul R. Desphande

    Present address: Department of Plant Biology, Michigan State University, East Lansing, MI, 48823, USA

  3. Heiko Hayen

    Present address: Department of Food Chemistry, University of Wuppertal, Gaußstr. 20, 42119, Wuppertal, Germany

Authors and Affiliations

  1. Laboratory of Chemical Biotechnology, Department of Biochemical and Chemical Engineering, TU Dortmund University, 44221, Dortmund, Germany

    Lars M. Blank, Rahul R. Desphande & Andreas Schmid

  2. Leibniz-Institut für Analytische Wissenschaften-ISAS-e.V, 44139, Dortmund, Germany

    Heiko Hayen

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  1. Lars M. Blank
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Correspondence to Heiko Hayen.

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Published in the special issue Young Investigators in Analytical and Bioanalytical Science with guest editors S. Daunert, J. Bettmer, T. Hasegawa, Q. Wang and Y. Wei.

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Blank, L.M., Desphande, R.R., Schmid, A. et al. Analysis of carbon and nitrogen co-metabolism in yeast by ultrahigh-resolution mass spectrometry applying 13C- and 15N-labeled substrates simultaneously. Anal Bioanal Chem 403, 2291–2305 (2012). https://doi.org/10.1007/s00216-012-6009-4

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  • DOI: https://doi.org/10.1007/s00216-012-6009-4

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