Metabolite isomers play diverse and crucial roles in various metabolic processes. However, in untargeted metabolomics analysis, it remains a great challenge to distinguish between the constitutional isomers and enantiomers of amine-containing metabolites due to their similar chemical structures and physicochemical properties. In this work, the triplex stable isotope N-phosphoryl amino acids labeling (SIPAL) is developed to identify and relatively quantify the amine-containing metabolites and their isomers by using chiral phosphorus reagents coupled with high-resolution tandem mass spectroscopy. The constitutional isomers could be effectively distinguished with stereo isomers by using the diagnosis ions in MS/MS spectra. The in-house software MS-Isomerism has been parallelly developed for high-throughput screening and quantification. The proposed strategy enables the unbiased detection and relative quantification of isomers of amine-containing metabolites. Based on the characteristic triplet peaks with SIPAL tags, a total of 854 feature peaks with 154 isomer groups are successfully recognized as amine-containing metabolites in liver cells, in which 37 amine-containing metabolites, including amino acids, polyamines, and small peptides, are found to be significantly different between liver cancer cells and normal cells. Notably, it is the first time to identify S-acetyl-glutathione as an endogenous metabolite in liver cells. The SIPAL strategy could provide spectacular insight into the chemical structures and biological functions of the fascinating amine-containing metabolite isomers. The feasibility of SIPAL in isomeric metabolomics analysis may reach a deeper understanding of the mirror-chemistry in life and further advance the discovery of novel biomarkers for disease diagnosis.

中文翻译：

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使用手性磷试剂全面分析含胺代谢物异构体

代谢异构体在各种代谢过程中发挥着多样化且至关重要的作用。然而，在非靶向代谢组学分析中，由于含胺代谢物的化学结构和理化性质相似，区分其结构异构体和对映异构体仍然是一个巨大的挑战。在这项工作中，开发了三重稳定同位素N-磷酰氨基酸标记（SIPAL），通过使用手性磷试剂与高分辨率串联质谱联用来识别和相对定量含胺代谢物及其异构体。利用MS/MS谱中的诊断离子可以有效区分结构异构体和立体异构体。同时开发了内部软件 MS-Isomerism，用于高通量筛选和定量。所提出的策略能够对含胺代谢物的异构体进行公正的检测和相对定量。基于带有SIPAL标签的特征三重峰，成功识别出154个异构体组的854个特征峰为肝细胞内的含胺代谢物，其中37个含胺代谢物，包括氨基酸、多胺和小肽，研究发现肝癌细胞与正常细胞之间存在显着差异。值得注意的是，这是首次将S-乙酰基谷胱甘肽鉴定为肝细胞内源性代谢物。SIPAL 策略可以为令人着迷的含胺代谢物异构体的化学结构和生物功能提供令人惊叹的见解。SIPAL在异构代谢组学分析中的可行性可能会加深对生命镜像化学的理解，并进一步推动疾病诊断的新型生物标志物的发现。