Skip to main content
SpringerLink
Log in

Determination of Isotopologue and Tandem Mass Isotopologue Ratios Using Gas Chromatography Chemical Ionization Time of Flight Mass Spectrometry - Methodology and Uncertainty of Measurement

  • Protocol
  • First Online:
Metabolic Flux Analysis in Eukaryotic Cells

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2088))

Abstract

The accurate and precise analysis of isotopologue and tandem mass isotopologue ratios in heavy stable isotope labeling experiments is a critical part of assessing absolute intracellular metabolic fluxes. Resulting from feeding the organism of interest with a specifically isotope-labeled substrate, the principal characteristics of these labeling experiments are the metabolites’ non-naturally distributed isotopologue patterns. For the purpose of inferring metabolic rates by maximizing the fit between a priori simulated and experimentally obtained labeling patterns, 13C is the preferred stable isotope of use.

The analysis of the obtained labeling patterns can be approached by different mass spectrometric approaches. Gas chromatography (GC) features broad metabolite coverage and excellent separation efficiency of biologically relevant isomers. These advantages compensate for laborious derivatization steps and the resulting need for interference correction for natural abundant isotopes.

Here, we describe a workflow based on GC-high resolution mass spectrometry with chemical ionization for the analysis of carbon-isotopologue distributions and some positional labeling information of primary metabolites. To study the associated measurement uncertainty of the resulting 13C labeling patterns, guidance to uncertainty estimation according to the EURACHEM guidelines with Monte-Carlo simulation is provided.

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

Access this chapter

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 89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 119.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
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

Institutional subscriptions

References

  1. Sauer U (2006) Metabolic networks in motion: 13C-based flux analysis. Mol Syst Biol 2:62. https://doi.org/10.1038/msb4100109

    Article  PubMed  PubMed Central  Google Scholar 

  2. Sauer U, Lasko DR, Fiaux J, Hochuli M, Glaser R, Szyperski T, Wuthrich K, Bailey JE (1999) Metabolic flux ratio analysis of genetic and environmental modulations of Escherichia coli central carbon metabolism. J Bacteriol 181:6679–6688

    CAS  PubMed  PubMed Central  Google Scholar 

  3. Wiechert W (2001) 13C metabolic flux analysis. Metab Eng 3:195–206. https://doi.org/10.1006/mben.2001.0187

    Article  CAS  PubMed  Google Scholar 

  4. Zamboni N (2011) 13C metabolic flux analysis in complex systems. Curr Opin Biotechnol 22:103–108. https://doi.org/10.1016/j.copbio.2010.08.009

    Article  CAS  PubMed  Google Scholar 

  5. Antoniewicz MR (2013) 13C metabolic flux analysis: optimal design of isotopic labeling experiments. Curr Opin Biotechnol 24:1116–1121. https://doi.org/10.1016/j.copbio.2013.02.003

    Article  CAS  PubMed  Google Scholar 

  6. McNaught AD, Wilkinson A (1997) IUPAC. Compendium of chemical terminology, 2nd edn. Blackwell Scientific Publications, Oxford

    Google Scholar 

  7. Kappelmann J, Klein B, Geilenkirchen P, Noack S (2017) Comprehensive and accurate tracking of carbon origin of LC-tandem mass spectrometry collisional fragments for 13C-MFA. Anal Bioanal Chem 409(9):2309–2326. https://doi.org/10.1007/s00216-016-0174-9

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. McCloskey D, Young JD, Xu S, Palsson BO, Feist AM (2016) MID max: LC–MS/MS method for measuring the precursor and product mass Isotopomer distributions of metabolic intermediates and cofactors for metabolic flux analysis applications. Anal Chem 88:1362–1370. https://doi.org/10.1021/acs.analchem.5b03887

    Article  CAS  PubMed  Google Scholar 

  9. Mairinger T, Hann S (2017) Implementation of data-dependent isotopologue fragmentation in 13C-based metabolic flux analysis. Anal Bioanal Chem 409:3713–3718. https://doi.org/10.1007/s00216-017-0339-1

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Zamboni N, Fischer E, Sauer U (2005) FiatFlux—a software for metabolic flux analysis from 13C-glucose experiments. BMC Bioinformatics 6:209. https://doi.org/10.1186/1471-2105-6-209

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Chu DB, Troyer C, Mairinger T, Ortmayr K, Neubauer S, Koellensperger G, Hann S (2015) Isotopologue analysis of sugar phosphates in yeast cell extracts by gas chromatography chemical ionization time-of-flight mass spectrometry. Anal Bioanal Chem 407:2865–2875. https://doi.org/10.1007/s00216-015-8521-9

    Article  CAS  PubMed  Google Scholar 

  12. Mairinger T, Steiger M, Nocon J, Mattanovich D, Koellensperger G, Hann S (2015) Gas chromatography-Quadrupole time-of-flight mass spectrometry-based determination of Isotopologue and tandem mass Isotopomer fractions of primary metabolites for 13C-metabolic flux analysis. Anal Chem 87:11792–11802. https://doi.org/10.1021/acs.analchem.5b03173

    Article  CAS  PubMed  Google Scholar 

  13. Van Winden WA, Wittmann C, Heinzle E, Heijnen JJ (2002) Correcting mass isotopomer distributions for naturally occurring isotopes. Biotechnol Bioeng 80:477–479. https://doi.org/10.1002/bit.10393

    Article  CAS  PubMed  Google Scholar 

  14. Millard P, Letisse F, Sokol S, Portais J-C (2012) IsoCor: correcting MS data in isotope labeling experiments. Bioinformatics 28:1294–1296. https://doi.org/10.1093/bioinformatics/bts127

    Article  CAS  PubMed  Google Scholar 

  15. Jungreuthmayer C, Neubauer S, Mairinger T, Zanghellini J, Hann S (2015) ICT: isotope correction toolbox. Bioinformatics 32(1):154–156. https://doi.org/10.1093/bioinformatics/btv514

    Article  CAS  PubMed  Google Scholar 

  16. Halket JM, Waterman D, Przyborowska AM, Patel RKP, Fraser PD, Bramley PM (2005) Chemical derivatization and mass spectral libraries in metabolic profiling by GC/MS and LC/MS/MS. J Exp Bot 56:219–243. https://doi.org/10.1093/jxb/eri069

    Article  CAS  PubMed  Google Scholar 

  17. 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 81:10038–10048. https://doi.org/10.1021/ac9019522

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Koek MM, Jellema RH, van der Greef J, Tas AC, Hankemeier T (2011) Quantitative metabolomics based on gas chromatography mass spectrometry: status and perspectives. Metabolomics 7:307–328. https://doi.org/10.1007/s11306-010-0254-3

    Article  CAS  PubMed  Google Scholar 

  19. Cipollina C, ten Pierick A, Canelas AB, Seifar RM, van Maris AJA, van Dam JC, Heijnen JJ (2009) A comprehensive method for the quantification of the non-oxidative pentose phosphate pathway intermediates in Saccharomyces cerevisiae by GC–IDMS. J Chromatogr B 877:3231–3236. https://doi.org/10.1016/j.jchromb.2009.07.019

    Article  CAS  Google Scholar 

  20. Harvey D, Horning M (1973) Characterization of the trimethylsilyl derivatives of sugar phosphates and related ompounds by gas chromatography and gas chromatography-mass spectrometry. J Chromatogr 76(1):51–62

    Article  CAS  PubMed  Google Scholar 

  21. Zamboni N, Fendt S-M, Rühl M, Sauer U (2009) 13C-based metabolic flux analysis. Nat Protoc 4:878–892. https://doi.org/10.1038/nprot.2009.58

    Article  CAS  PubMed  Google Scholar 

  22. Mairinger T, Wegscheider W, Peña DA, Steiger MG, Koellensperger G, Zanghellini J, Hann S (2018) Comprehensive assessment of measurement uncertainty in 13C-based metabolic flux experiments. Anal Bioanal Chem 410:3337–3348. https://doi.org/10.1007/s00216-018-1017-7

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Wahl SA, Dauner M, Wiechert W (2004) New tools for mass isotopomer data evaluation in 13C flux analysis: mass isotope correction, data consistency checking, and precursor relationships. Biotechnol Bioeng 85:259–268. https://doi.org/10.1002/bit.10909

    Article  CAS  PubMed  Google Scholar 

  24. Moseley HN (2010) Correcting for the effects of natural abundance in stable isotope resolved metabolomics experiments involving ultra-high resolution mass spectrometry. BMC Bioinformatics 11:139. https://doi.org/10.1186/1471-2105-11-139

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Niedenführ S, ten Pierick A, van Dam PTN, Suarez-Mendez CA, Nöh K, Wahl SA (2016) Natural isotope correction of MS/MS measurements for metabolomics and 13C fluxomics. Biotechnol Bioeng 113:1137–1147. https://doi.org/10.1002/bit.25859

    Article  CAS  PubMed  Google Scholar 

  26. BIPM (2012) International vocabulary of metrology—basic and general concepts and associated terms (VIM). BIPM, Sèvres

    Google Scholar 

  27. Ellison S, Williams A (2012) Eurachem/CITAC guide: quantifying uncertainty in analytical measurement, 3rd edn. LGC (Teddington) Limited, Teddington

    Google Scholar 

  28. Millard P, Massou S, Portais J-C, Létisse F (2014) Isotopic studies of metabolic systems by mass spectrometry: using Pascal’s triangle to produce biological standards with fully controlled labeling patterns. Anal Chem 86:10288–10295. https://doi.org/10.1021/ac502490g

    Article  CAS  PubMed  Google Scholar 

  29. Russmayer H, Troyer C, Neubauer S, Steiger MG, Gasser B, Hann S, Koellensperger G, Sauer M, Mattanovich D (2015) Metabolomics sampling of Pichia pastoris revisited: rapid filtration prevents metabolite loss during quenching. FEMS Yeast Res 15:fov049. https://doi.org/10.1093/femsyr/fov049

    Article  CAS  PubMed  Google Scholar 

  30. van Gulik WM (2010) Fast sampling for quantitative microbial metabolomics. Curr Opin Biotechnol 21:27–34. https://doi.org/10.1016/j.copbio.2010.01.008

    Article  CAS  PubMed  Google Scholar 

  31. Hernández Bort JA, Shanmukam V, Pabst M, Windwarder M, Neumann L, Alchalabi A, Krebiehl G, Koellensperger G, Hann S, Sonntag D, Altmann F, Heel C, Borth N (2014) Reduced quenching and extraction time for mammalian cells using filtration and syringe extraction. J Biotechnol 182–183(1):97–103

    Article  PubMed  PubMed Central  Google Scholar 

  32. Koek MM, Muilwijk B, van der Werf MJ, Hankemeier T (2006) Microbial metabolomics with gas chromatography/mass spectrometry. Anal Chem 78:1272–1281

    Article  CAS  PubMed  Google Scholar 

  33. Vielhauer O, Zakhartsev M, Horn T, Takors R, Reuss M (2011) Simplified absolute metabolite quantification by gas chromatography–isotope dilution mass spectrometry on the basis of commercially available source material. J Chromatogr B 879:3859–3870. https://doi.org/10.1016/j.jchromb.2011.10.036

    Article  CAS  Google Scholar 

  34. Berglund M, Wieser ME (2011) Isotopic compositions of the elements 2009 (IUPAC technical report). Pure Appl Chem 83:397–410. https://doi.org/10.1351/PAC-REP-10-06-02

    Article  CAS  Google Scholar 

  35. Kragten J (1994) Tutorial review. Calculating standard deviations and confidence intervals with a universally applicable spreadsheet technique. Analyst 119:2161–2165. https://doi.org/10.1039/AN9941902161

    Article  CAS  Google Scholar 

Download references

Author information

Author notes

  1. Teresa Mairinger

    Present address: Department of Environmental Chemistry, Eawag: Swiss Federal Institute of Aquatic Science and Technology, Duebendorf, Switzerland

Authors and Affiliations

  1. Department of Chemistry, University of Natural Resources and Life Sciences—BOKU Vienna, Vienna, Austria

    Teresa Mairinger & Stephen Hann

  2. Austrian Center for Industrial Biotechnology, Vienna, Austria

    Stephen Hann

Authors
  1. Teresa Mairinger
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Stephen Hann
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Teresa Mairinger .

Editor information

Editors and Affiliations

  1. Department of Biomedical Engineering, University of Michigan, Ann Arbor, MI, USA

    Deepak Nagrath

Rights and permissions

Reprints and permissions

Copyright information

© 2020 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Mairinger, T., Hann, S. (2020). Determination of Isotopologue and Tandem Mass Isotopologue Ratios Using Gas Chromatography Chemical Ionization Time of Flight Mass Spectrometry - Methodology and Uncertainty of Measurement. In: Nagrath, D. (eds) Metabolic Flux Analysis in Eukaryotic Cells. Methods in Molecular Biology, vol 2088. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0159-4_1

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-0159-4_1

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-0158-7

  • Online ISBN: 978-1-0716-0159-4

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation