Unbiased high-throughput screening of reactive metabolites on the linear ion trap mass spectrometer using polarity switch and mass tag triggered data-dependent acquisition.
Unbiased high-throughput screening of reactive metabolites on the linear ion trap mass spectrometer using polarity switch and mass tag triggered data-dependent acquisition.
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DOI：
10.1021/ac800887h
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发表时间：
2008-07
影响因子：
7.4
通讯作者：
Zhengyin Yan;G. Caldwell;Noureddine Maher
中科院分区：
文献类型：
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作者：
Zhengyin Yan;G. Caldwell;Noureddine Maher
恒定中性丢失（CNL）和前体离子（PI）扫描已被广泛用于活性代谢产物谷胱甘肽结合物的体外筛选，但这两种方法仅适用于三重四极杆或混合三重四极杆质谱仪。此外，CNL和PI扫描的成功很大程度上取决于GSH缀合物的结构和CID片段化途径。在本研究中，一个高效的方法已被开发作为一种替代方法，高通量筛选和结构表征的活性代谢物使用线性离子阱质谱仪。在微粒体孵育中，使用谷胱甘肽[GSH，γ-谷氨酰-半胱氨酸-甘氨酸]和稳定同位素标记化合物[GSX，γ-谷氨酰-半胱氨酸-甘氨酸-（13）C2-（15）N]的混合物捕获反应性代谢物，从而形成给定同位素比的标记和未标记结合物。在固定的同位素比例下，天然和标记的GSH结合物（质量标签）之间的质量差为3.0 Da，这构成了一种独特的质量模式，可以分别选择性地触发两种同位素伴侣离子的数据依赖性MS（2）扫描。为了消除GSH加合物在正和负模式下的响应偏倚，在质量标签触发的数据依赖性MS（2）扫描之间执行极性切换，从而在单次LC-MS运行中获得标记和未标记GSH结合物的ESI-和ESI+ MS（2）光谱。通过标记和未标记结合物的MS（2）光谱，可以很容易地实现谷胱甘肽加合物的非环境生物识别，具有很大的置信度。使用已知可形成反应性代谢物的几种模型化合物，对该方法的可靠性进行了有力验证。该方法不是基于特定产物离子的出现，例如MH（+）-129和m/z 272处的阴离子，其形成可能是结构依赖性的并且对碰撞能级敏感;因此，本方法可适用于任何反应性代谢物的无偏筛选，而不管其CID裂解途径如何。此外，该方法可以潜在地应用于三重四极杆或混合三重四极杆质谱仪。
Constant neutral loss (CNL) and precursor ion (PI) scan have been widely used for the in vitro screening of glutathione conjugates derived from reactive metabolites, but these two methods are only applicable to triple quadrupole or hybrid triple quadrupole mass spectrometers. Additionally, the success of CNL and PI scanning largely depends on structure and CID fragmentation pathways of GSH conjugates. In the present study, a highly efficient methodology has been developed as an alternative approach for high-throughput screening and structural characterization of reactive metabolites using the linear ion trap mass spectrometer. In microsomal incubations, a mixture of glutathione [GSH, gamma-glutamyl-cystein-glycin] and the stable-isotope labeled compound [GSX, gamma-glutamyl-cystein-glycin-(13)C2-(15)N] was used to trap reactive metabolites, resulting in formation of both labeled and unlabeled conjugates at a given isotopic ratio. A mass difference of 3.0 Da between the natural and labeled GSH conjugate (mass tag) at a fixed isotopic ratio constitutes a unique mass pattern that can selectively trigger the data-dependent MS(2) scan of both isotopic partner ions, respectively. In order to eliminate the response bias of GSH adducts in the positive and negative mode, a polarity switch is executed between the mass tag-triggered data dependent MS(2) scan, and thus ESI- and ESI+ MS(2) spectra of both labeled and nonlabeled GSH conjugates are obtained in a single LC-MS run. Unambiguous identification of glutathione adducts was readily achieved with great confidence by MS(2) spectra of both labeled and unlabeled conjugates. Reliability of this method was vigorously validated using several model compounds that are known to form reactive metabolites. This approach is not based on the appearance of a particular product ion such as MH(+) - 129 and anion at m/z 272, whose formation can be structure-dependent and sensitive to the collision energy level; therefore, the present method can be suitable for unbiased screening of any reactive metabolites, regardless of their CID fragmentation pathways. Additionally, this methodology can potentially be applied to triple quadrupole or hybrid triple quadrupole mass spectrometers.