A single-vial analytical and quantitative gas chromatography-mass spectrometry assay for terpene synthases.
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.
O'Maille
Paul E
PE
Structural Biology Laboratory, Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, CA 92037, USA.
Chappell
Joe
J
Noel
Joseph P
JP
D019883:Alkyl and Aryl Transferases / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D019755:Carbon-Carbon Lyases / Q000378:metabolism; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods*; D012717:Sesquiterpenes / Q000737:chemistry; D013729:Terpenes / Q000737:chemistry*; D014026:Nicotiana
D012717:Sesquiterpenes; D013729:Terpenes; D019883:Alkyl and Aryl Transferases; C078217:5-epi-aristolochene synthase; C403714:amorpha-4,11-diene synthase; C108877:terpene synthase; D019755:Carbon-Carbon Lyases; C051130:trichodiene synthetase