A single-vial analytical and quantitative gas chromatography-mass spectrometry assay for terpene synthases
A single-vial analytical and quantitative gas chromatography-mass spectrometry assay for terpene synthases
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DOI：
10.1016/j.ab.2004.09.011
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发表时间：
2004-12-15
影响因子：
2.9
通讯作者：
Noel, JP
中科院分区：
文献类型：
--
作者：
O'Maille, PE;Chappell, J;Noel, JP
开发了用于分析倍半萜烯脱氢酶的定量测定，其中将每种反应混合物配制在玻璃气相色谱小瓶中，用有机溶剂如乙酸乙酯覆盖，随后涡旋以在合适的温育期后将烃反应产物萃取到有机相中。然后以自动化方式对产物富集的有机相进行取样，并直接注入气相色谱-质谱仪中，而无需进一步后处理以分析和定量烃产物。应用小瓶分析紫穗槐-4，11-二烯合酶（ADS），倍半萜合酶的分析，证明了灵敏度的检测主要和次要反应产物的测定，最值得注意的是几个倍半萜产品的识别，已经逃脱了以前的检测。烟草5-epi-aristolochene合酶（TEAS），另一种倍半萜合酶的稳态动力学分析，验证了定量性质的测定，提供了一种替代手段，建立的方法，使用放射性标记的底物，提取，和闪烁计数。这种简化的分析提供了一种标准化的方法，以促进萜烯脱氢酶和不同的突变体酶库的分析，取代了在气相色谱-质谱（GG-MS）分析之前使用较大规模反应、多次萃取和有机相蒸发浓缩的常见做法。(C)2004年爱思唯尔公司All rights reserved.
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 (GG-MS) analysis. (C) 2004 Elsevier Inc. All rights reserved.