A single-vial analytical and quantitative gas chromatography-mass spectrometry assay for terpene synthases

Paul E. O'Maille, Joe Chappell, Joseph P. Noel

Research output: Contribution to journalArticlepeer-review

56 Scopus citations

Abstract

A quantitative assay for the analysis of sesquiterpene synthases, wherein each reaction mixture is formulated in glass gas chromatography vials, overlaid with organic solvent such as ethyl acetate, and subsequently vortexed to extract hydrocarbon reaction products into the organic phase after a suitable incubation period, was developed. The product-enriched organic phase is then sampled in an automated fashion and injected directly into a gas chromatograph-mass spectrometer without further workup for analysis and quantification of hydrocarbon products. Application of the vial assay to the analysis of amorpha-4,11-diene synthase (ADS), a sesquiterpene synthase, demonstrated the sensitivity of the assay for detection of major and minor reaction products and most notably for the identification of several sesquiterpene products that had escaped previous detection. A steady-state kinetic analysis of tobacco 5-epi-aristolochene synthase (TEAS), another sesquiterpene synthase, validated the quantitative nature of the assay, providing an alternative means to the established method of using radiolabeled substrate, extraction, and scintillation counting. This simplified assay provides a standardized method to facilitate analysis of terpene synthases and diverse mutant enzyme libraries by supplanting the common practice of using larger scale reactions, multiple extractions, and evaporative concentration of the organic phase prior to gas chromatography-mass spectrometry (GC-MS) analysis.

Original languageEnglish
Pages (from-to)210-217
Number of pages8
JournalAnalytical Biochemistry
Volume335
Issue number2
DOIs
StatePublished - Dec 15 2004

Bibliographical note

Funding Information:
We thank Peter Brodelius for the ADS clone used in these studies. We are grateful to the National Institutes of health for grants that supported this work (GM54029 to Joseph P. Noel and Joe Chappell). Paul E. O’Maille is an NIH Postdoctoral Research Fellow (GM069056-01).

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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