Stable-isotope trapping and high-throughput screenings of reactive metabolites using the isotope MS signature
Stable-isotope trapping and high-throughput screenings of reactive metabolites using the isotope MS signature
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DOI：
10.1021/ac040159k
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发表时间：
2004-12-01
影响因子：
7.4
通讯作者：
Caldwell, GW
中科院分区：
文献类型：
--
作者：
Yan, ZY;Caldwell, GW
利用稳定同位素捕集结合电喷雾质谱中性丢失扫描，建立了一种高效的活性代谢物检测和鉴定方法。以等摩尔比使用谷胱甘肽（GSH，γ-谷氨酰半胱氨酰甘氨酸）和稳定同位素标记化合物（GSX，γ-谷氨酰半胱氨酰甘氨酸-C-13（2）-N-15）的混合物来捕获微粒体孵育中产生的反应性代谢物。通过SPE清洁并浓缩孵育所得样品，然后使用129 Da（γ-谷氨酰部分）的恒定中性丢失扫描进行LC-MS/MS分析，以检测形成的GSH结合物。谷胱甘肽加合物的Unambibibility鉴定大大促进了一个突出的同位素双峰，不同的质量由3 Da. Further的结构表征的共轭物的存在下实现了高置信度，随后获得的MS/MS光谱，其特点是中性损失75和129 Da的GSH加合物和78和129 Da的同位素GSX加合物。使用许多已知可形成反应性代谢物的化合物对该方法的可靠性进行了有力验证。本方法鉴定低丰度活性代谢物的能力证明了上级灵敏度。由于独特的同位素MS特征，反应性代谢物的超快分析是通过将清洁的样品直接注入质谱仪进行中性损失扫描来完成的。更重要的是，这项研究已经证明了通过计算机辅助模式识别完全自动化检测反应性代谢物的当前方法的可行性。
A highly efficient method has been developed to detect and identify reactive metabolites, using stable-isotope trapping combined with ESI-MS/MS neutral loss scanning. A mixture of glutathione (GSH, gamma-glutamylcysteinylglycine) and the stable-isotope labeled compound (GSX, gamma-glutamylcysteinylglycine-C-13(2)-N-15) was used at an equal molar ratio to trap reactive metabolites generated in microsomal incubations. Samples resulting from incubations were cleaned and concentrated by SPE, followed by LC-MS/MS analyses using constant neutral loss scanning for 129 Da (the gamma-glutamyl moiety) to detect formed GSH conjugates. Unambiguous identification of glutathione adducts was greatly facilitated by the presence of a unique MS signature of a prominent isotopic doublet that differs in mass by 3 Da. Further structural characterization of conjugates was achieved with high confidence by subsequently acquiring MS/MS spectra that were featured by neutral losses of 75 and 129 Da for GSH adducts and 78 and 129 Da for isotopic GSX adducts. The reliability of this method was vigorously validated using a number of compounds known to form reactive metabolites. Superior sensitivity was demonstrated by the capability of the current approach to identify reactive metabolites at low abundance. Because of the unique isotopic MS signature, ultrafast analyses of reactive metabolites were accomplished by direct injection of cleaned samples into mass spectrometers for neutral loss scanning. More importantly, this study has demonstrated the feasibility of the current method for completely automated detection of reactive metabolites via computer-assisted pattern recognition.