对于外源性生物代谢的分析,代谢物通常通过高分辨率质谱鉴定,如轨道阱或飞行时间质谱仪,并通过基于三重四极杆(QQQ)质谱仪的多反应监测进行定量。虽然此工作流程在仪器参数传输困难方面显示出缺点,和QQQ提供较少的特异性。在这项工作中,我们构建了一种基于高分辨率MS/MS(HR-MS/MS)的策略,通过代谢途径延伸(MPE)搜索与平行反应监测(PRM)相结合,提高了未知外源性代谢物的发现和定量.以糖尿病创面S9孵育液中的黄酮代谢为试验案例。首先,MPE方法用于筛选所有潜在的代谢物。在这一步,通过21种常见的异生代谢反应,基于预定义的类黄酮结构构建了所有理论类黄酮代谢产物的m/z值库,并且该库与从黄酮-S9共孵育的原始数据(MS1扫描)中提取的所有特征相匹配,然后将匹配的特征导出到MS2碎片的目标列表中以进行结构验证。其次,代谢物根据其特征产物离子通过PRM模式进行相对定量。因此,鉴定了皮肤和肌肉中9种不同黄酮类化合物的131种代谢产物。据我们所知,这是关于皮肤或肌肉组织中类黄酮代谢的首次报道。结果还验证了所提出的基于HR-MS/MS的策略在未知的异源代谢物的发现和定量过程中提供了高特异性,而无需仪器参数传递。
For the analysis of xenobiotic metabolism, metabolites are commonly qualified by high-resolution mass spectrometry such as orbitrap or time-of-flight mass spectrometers, and quantified by triple-quadrupole (QQQ) mass spectrometer based multiple reaction monitoring. While this workflow shows drawback in the difficulty for instrumental parameters transfer, and QQQ provides less specificity. In this work, we constructed a high-resolution MS/MS (HR-MS/MS) based strategy to improve the discovery and quantification of unknown xenobiotic metabolites by metabolic pathway extension (MPE) searching combined with parallel reaction monitoring (PRM). Taking the
flavonoid metabolism in diabetes wound S9 incubates as a test
case. Firstly, MPE approach was used to screen all potential metabolites. In this step, an m/z value library of all theoretic
flavonoid metabolites were constructed based on predefined
flavonoid structures through 21 common xenobiotic metabolic reactions, and this library was matched with all features extracted from raw data (MS1 scan) of
flavonoid-S9 co-incubate, then the matched features were exported into target list for MS2 fragmentation for structure validation. Secondly, the metabolites were relatively quantified by PRM mode based on their characteristic product ions. As a result, 131 metabolites of 9 different kinds of flavonoids in the skin and muscle were identified. To our best knowledge, this is the first report on the metabolism of flavonoids in the skin or muscle tissue. The results also validated the proposed HR-MS/MS-based strategy provided high specificity throughout both discovery and quantitation process of unknown xenobiotic metabolites without need of instrumental parameter transfer.