Biotechnology Letters

, Volume 39, Issue 12, pp 1853–1857 | Cite as

Measurement of the 15N/14N ratio of phenylalanine in fermentation matrix by isotope ratio mass spectrometry

  • Shiwei Zhang
  • Qiding Zhong
  • Daobing Wang
  • Zhanbin Huang
  • Guohui Li
Original Research Paper
  • 114 Downloads

Abstract

Objectives

To determine the origin of 15N-labeled phenylalanine in microbial metabolic flux analysis using 15N as a tracer, a method for measuring phenylalanine δ15N using HPLC coupled with elemental analysis-isotope ratio mass spectrometry (EA-IRMS) was developed.

Results

The original source of the 15N-labeled phenylalanine was determined using this new method that consists of three steps: optimization of the HPLC conditions, evaluation of the isotope fractionation effects, and evaluation of the effect of pre-processing on the phenylalanine nitrogen stable isotope. In addition, the use of a 15N-labeled inorganic nitrogen source, rather than 15N-labeled amino acids, was explored using this method.

Conclusions

The method described here can also be applied to the analysis of metabolic flux.

Keywords

Elemental analysis-isotope ratio mass spectrometry (EA-IRMS) High-performance liquid chromatography Metabolic flux analysis Phenylalanine δ15

Notes

Acknowledgements

This study was supported by the International S&T Cooperation Program of China (2015DFA31720), the National Nature Foundation of China (31601580) and the National Science and Technology Project of the Ministry of Science and Technology in the 13th Five-Year Plan Period (2016YFF0203903).

Supporting information

Supplementary Fig. 1—Preparative separation of microbial hydrolysis mixture.

Supplementary Fig. 2—Measured δ15N value of phenylalanine added with phenylalanine standard material.

Supplementary Fig. 3—15N phenylalanine enrichment is in linear relationship with 15N-labeled NH4Cl concentration.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

10529_2017_2387_MOESM1_ESM.docx (132 kb)
Supplementary material 1 (DOCX 131 kb)

References

  1. Bart V, Middelburg JJ, Boschker HTS, Houtekamer M (2005) Analysis of 15N incorporation into d-alanine: a new method for tracing nitrogen uptake by bacteria. Limnol Oceanog Meth 3:230–240CrossRefGoogle Scholar
  2. Blank LM, Desphande RR, Schmid A (2012) Analysis of carbon and nitrogen co-metabolism in yeast by ultrahigh-resolution mass spectrometry applying 13C- and 15N-labeled substrates simultaneously. Anal Bioanal Chem 403:2291–2305CrossRefPubMedGoogle Scholar
  3. Chikaraishi Y, Kashiyama Y, Ogawa NO, Kitazato H, Ohkouchi N (2007) Metabolic control of nitrogen isotope composition of amino acids in macroalgae and gastropods: implications for aquatic food web studies. Mar Ecol Prog Ser 342:85–90CrossRefGoogle Scholar
  4. Henrik M, Giovanni PA, Akhilesh P (2008) Assessing reproducibility of a protein dynamics study using in vivo labeling and liquid chromatography tandem mass spectrometry. Anal Chem 77:2739–2744Google Scholar
  5. Ippel JH, Pouvreau L, Kroef T (2004) In vivo uniform 15N-isotope labelling of plants: using the greenhouse for structural proteomics. Proteomics 4:226–234CrossRefPubMedGoogle Scholar
  6. McCarthy MD, Benner R, Lee C, Fogel ML (2007) Amino acid nitrogen isotopic fractionation patterns as indicators of heterotrophy in plankton, particulate, and dissolved organic matter. Geochim Cosmochim Acta 71:4727–4744CrossRefGoogle Scholar
  7. McClelland JW, Montoya JP (2002) Trophic relationships and the nitrogen isotopic composition of amino acids in plankton. Ecology 83:2173–2180CrossRefGoogle Scholar
  8. Nelson ST (2000) A simple, practical methodology for routine VSMOW/SLAP normalization of water samples analyzed by continuous flow methods. Rapid Commun Mass Spectrom 14:1044–1046CrossRefPubMedGoogle Scholar
  9. Piasentiera E, Valussoa R, Caminb F, Versinib G (2003) Stable isotope ratio analysis for authentication of lamb meat. Meat Sci 64:239–247CrossRefGoogle Scholar
  10. Schmidt O, Quilter JM, Bahar B, Moloney AP, Scrimgeour CM, Begley IS, Monahan FJ (2005) Inferring the origin and dietary history of beef from C, N and S stable isotope ratio analysis. Food Chem 91:545–549CrossRefGoogle Scholar
  11. Smet KD, Contreras R (2005) Human antimicrobial peptides: defensins, cathelicidins and histatins. Biotechnol Lett 27:1337–1347CrossRefPubMedGoogle Scholar
  12. Tadakatsu Y, Kenzo K, Kikuo K (1975) Nitrogen transport in intact corn roots. Soil Sci Plant Nutr 21:371–377CrossRefGoogle Scholar
  13. Tripp JA, Mccullagh JSO, Hedges REM (2006) Preparative separation of underivatized amino acids for compound-specific stable isotope analysis and radiocarbon dating of hydrolyzed bone collagen. J Sep Sci 29:41–48CrossRefPubMedGoogle Scholar
  14. Tsuchida T, Kubota K, Morinaga Y, Matsui H, Enei H, Yoshinaga F (1987) Production of l-Phenylalanine by a mutant of Brevibacterium lactofermentum 2256. Agric Biol Chem 51:2095–2101Google Scholar

Copyright information

© Springer Science+Business Media B.V. 2017

Authors and Affiliations

  • Shiwei Zhang
    • 1
    • 2
    • 3
  • Qiding Zhong
    • 2
    • 3
  • Daobing Wang
    • 2
    • 3
  • Zhanbin Huang
    • 1
  • Guohui Li
    • 2
    • 3
  1. 1.School of Chemical & Environmental EngineeringChina University of Mining and Technology (Beijing)BeijingChina
  2. 2.China National Institute of Food and Fermentation IndustriesBeijingChina
  3. 3.National Standardization Center of Food & Fermentation IndustryBeijingChina

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