Skip to main content
SpringerLink
Log in

GC-MS/MS survey of collision-induced dissociation of tert-butyldimethylsilyl-derivatized amino acids and its application to 13C-metabolic flux analysis of Escherichia coli central metabolism

  • Research Paper
  • Published:
Analytical and Bioanalytical Chemistry Aims and scope Submit manuscript

Abstract

Stable isotope labeling experiments using mass spectrometry have been employed to investigate carbon flow levels (metabolic flux) in mammalian, plant, and microbial cells. To achieve a more precise 13C-metabolic flux analysis (13C-MFA), novel fragmentations of tert-butyldimethylsilyl (TBDMS)-amino acids were investigated by gas chromatography-tandem mass spectrometry (GC-MS/MS). The product ion scan analyses of 15 TBDMS-amino acids revealed 24 novel fragment ions. The amino acid-derived carbons included in the five fragment ions were identified by the analyses of 13C-labeled authentic standards. The identification of the fragment ion at m/z 170 indicated that the isotopic abundance of S-methyl carbon in methionine could be determined from the cleavage of C5 in the precursor of [M–159]+ (m/z 218). It was also confirmed that the precision of 13C-MFA in Escherichia coli central carbon metabolism could be improved by introducing 13C-labeling data derived from novel fragmentations.

Novel collision-induced dissociation fragmentations of tert-butyldimethylsilyl amino acids were investigated and identified by GC-MS/MS

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

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

Abbreviations

3PG:

3-Phosphoglycerate

6PG:

6-Phosphogluconate

AcCoA:

Acetyl-coenzyme A

Ala:

Alanine

Asp:

Aspartate

Cit:

Citrate

DHAP:

Dihydroxyacetone phosphate

E4P:

Erythrose 4-phosphate

F6P:

Fructose 6-phosphate

FBP:

Fructose 1,6-bisphosphate

Fum:

Fumarate

G6P:

Glucose 6-phosphate

GAP:

Glyceraldehyde 3-phosphate

Glu:

Glutamate

Gly:

Glycine

His:

Histidine

Ile:

Isoleucine

Leu:

Leucine

Lys:

Lysine

Mal:

Malate

Met:

Methionine

Oxa:

Oxaloacetate

PEP:

Phosphoenolpyruvate

Phe:

Phenylalanine

Pro:

Proline

Pyr:

Pyruvate

R5P:

Ribose 5-phosphate

Ru5P:

Ribulose 5-phosphate

S7P:

Sedoheptulose 7-phosphate

Ser:

Serine

Suc:

Succinate

Thr:

Threonine

Tyr:

Tyrosine

Val:

Valine

Xu5P:

Xylulose 5-phosphate

αKG:

α-Ketoglutarate

References

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

    Article  CAS  Google Scholar 

  2. Antoniewicz MR. Using multiple tracers for 13C metabolic flux analysis. Methods Mol Biol. 2013;985:353–65.

    Article  CAS  Google Scholar 

  3. Antoniewicz MR. Methods and advances in metabolic flux analysis: a mini-review. J Ind Microbiol Biotechnol. 2015;42(3):317–25.

    Article  CAS  Google Scholar 

  4. Wittmann C. Fluxome analysis using GC-MS. Microb Cell Factories. 2007;6:6.

    Article  Google Scholar 

  5. Zamboni N, Fendt SM, Ruhl M, Sauer U. 13C-based metabolic flux analysis. Nat Protoc. 2009;4(6):878–92.

    Article  CAS  Google Scholar 

  6. Dauner M, Sauer U. GC-MS analysis of amino acids rapidly provides rich information for isotopomer balancing. Biotechnol Prog. 2000;16(4):642–9.

    Article  CAS  Google Scholar 

  7. Antoniewicz MR, Kelleher JK, Stephanopoulos G. Accurate assessment of amino acid mass isotopomer distributions for metabolic flux analysis. Anal Chem. 2007;79(19):7554–9.

    Article  CAS  Google Scholar 

  8. Choi J, Antoniewicz MR. Tandem mass spectrometry: a novel approach for metabolic flux analysis. Metab Eng. 2011;13(2):225–33.

    Article  CAS  Google Scholar 

  9. Antoniewicz MR. Tandem mass spectrometry for measuring stable-isotope labeling. Curr Opin Biotechnol. 2013;24(1):48–53.

    Article  CAS  Google Scholar 

  10. Ruhl M, Rupp B, Noh K, Wiechert W, Sauer U, Zamboni N. Collisional fragmentation of central carbon metabolites in LC-MS/MS increases precision of 13C metabolic flux analysis. Biotechnol Bioeng. 2012;109(3):763–71.

    Article  Google Scholar 

  11. McCloskey D, Young JD, Xu S, Palsson BO, Feist AM. 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. 2016;88(2):1362–70.

    Article  CAS  Google Scholar 

  12. Niedenfuhr S, Pierick AT, van Dam PT, Suarez-Mendez CA, Noh K, Wahl SA. Natural isotope correction of MS/MS measurements for metabolomics and 13C fluxomics. Biotechnol Bioeng. 2015;113(5):1137–47.

    Article  Google Scholar 

  13. Choi J, Grossbach MT, Antoniewicz MR. Measuring complete isotopomer distribution of aspartate using gas chromatography/tandem mass spectrometry. Anal Chem. 2012;84(10):4628–32.

    Article  CAS  Google Scholar 

  14. Okahashi N, Kajihata S, Furusawa C, Shimizu H. Reliable metabolic flux estimation in Escherichia coli central carbon metabolism using intracellular free amino acids. Metabolites. 2014;4(2):408–20.

    Article  Google Scholar 

  15. Antoniewicz MR, Kraynie DF, Laffend LA, Gonzalez-Lergier J, Kelleher JK, Stephanopoulos G. Metabolic flux analysis in a nonstationary system: fed-batch fermentation of a high yielding strain of E. coli producing 1,3-propanediol. Metab Eng. 2007;9(3):277–92.

    Article  CAS  Google Scholar 

  16. van Winden WA, Wittmann C, Heinzle E, Heijnen JJ. Correcting mass isotopomer distributions for naturally occurring isotopes. Biotechnol Bioeng. 2002;80(4):477–9.

    Article  Google Scholar 

  17. Kajihata S, Furusawa C, Matsuda F, Shimizu H. OpenMebius: an open source software for isotopically nonstationary 13C-based metabolic flux analysis. BioMed Res Int. 2014;2014:627014.

    Article  Google Scholar 

  18. Antoniewicz MR, Kelleher JK, Stephanopoulos G. Elementary metabolite units (EMU): a novel framework for modeling isotopic distributions. Metab Eng. 2007;9(1):68–86.

    Article  CAS  Google Scholar 

  19. Crown SB, Long CP, Antoniewicz MR. Integrated 13C-metabolic flux analysis of 14 parallel labeling experiments in Escherichia coli. Metab Eng. 2015;28:151–8.

    Article  CAS  Google Scholar 

  20. Wasylenko TM, Stephanopoulos G. Kinetic isotope effects significantly influence intracellular metabolite 13C labeling patterns and flux determination. Biotechnol J. 2013;8(9):1080–9.

    Article  CAS  Google Scholar 

  21. Antoniewicz MR, Kelleher JK, Stephanopoulos G. Determination of confidence intervals of metabolic fluxes estimated from stable isotope measurements. Metab Eng. 2006;8(4):324–37.

    Article  CAS  Google Scholar 

  22. Toya Y, Ishii N, Nakahigashi K, Hirasawa T, Soga T, Tomita M. 13C-metabolic flux analysis for batch culture of Escherichia coli and its Pyk and Pgi gene knockout mutants based on mass isotopomer distribution of intracellular metabolites. Biotechnol Prog. 2010;26(4):975–92.

    CAS  Google Scholar 

  23. Antoniewicz MR. 13C metabolic flux analysis: optimal design of isotopic labeling experiments. Curr Opin Biotechnol. 2013;24(6):1116–21.

    Article  CAS  Google Scholar 

Download references

Acknowledgments

We thank Prof. Eiichiro Fukusaki and Prof. Yoshihiro Toya (Osaka University, Japan) for their helpful comments. This research was partially supported by JST, Strategic International Collaborative Research Program, SICORP, for JP-US Metabolomics.

Author information

Authors and Affiliations

  1. Department of Bioinformatic Engineering, Graduate School of Information Science and Technology, Osaka University, 1-5 Yamadaoka, Suita, Osaka, 565-0871, Japan

    Nobuyuki Okahashi, Hiroshi Shimizu & Fumio Matsuda

  2. Analytical and Measuring Instruments Division, Shimadzu Corporation, 1 Nishinokyo Kuwabara-cho, Nakagyo-ku, Kyoto, 604-8511, Japan

    Shuichi Kawana & Junko Iida

  3. Osaka University Shimadzu Analytical Innovation Research Laboratory, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka, 565-0871, Japan

    Junko Iida

Authors
  1. Nobuyuki Okahashi
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Shuichi Kawana
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Junko Iida
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Hiroshi Shimizu
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Fumio Matsuda
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Fumio Matsuda.

Ethics declarations

All remaining authors have declared no conflicts of interest.

Electronic supplementary material

Below is the link to the electronic supplementary material.

ESM 1

(PDF 331 kb)

ESM 2

(XLSX 71 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Okahashi, N., Kawana, S., Iida, J. et al. GC-MS/MS survey of collision-induced dissociation of tert-butyldimethylsilyl-derivatized amino acids and its application to 13C-metabolic flux analysis of Escherichia coli central metabolism. Anal Bioanal Chem 408, 6133–6140 (2016). https://doi.org/10.1007/s00216-016-9724-4

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00216-016-9724-4

Keywords

Search

Navigation