甲羟戊酸(MVA)通路在多种疾病的发生和发展中起着至关重要的作用,比如骨质疏松症,乳腺癌,肺癌,等。然而,由于它们的高极性,确定所有MVA途径中间体仍然具有挑战性,低浓度,与金属隔室的螯合作用,和较差的质谱响应。在这项研究中,我们建立了液相色谱-串联质谱(LC-MS/MS)结合N2,N2,N4,N4-四甲基-6-(4-(哌嗪-1-基磺酰基)苯基)-1,3,5-三嗪-2,4-二胺(Tmt-PP)标记的方法,用于同时分析生物样品中所有MVA中间体。化学衍生显著提高了色谱保留率,峰形,和分析物的检测灵敏度。此外,我们采用了一种名为质谱计算的方法来实现异构体的绝对定量,即,异戊烯焦磷酸(IPP)和二甲基烯丙基焦磷酸(DMAPP)。建立的方法完全合格,并应用于探索这些代谢物在顺铂耐药的非小细胞肺癌(NSCLC)细胞中的差异。此外,几种MVA中间类似物,包括单磷酸异戊烯酯或单磷酸二甲基烯丙基酯(IMP/DMAMP),一磷酸香叶酯(GMP),5-三磷酸甲羟戊酸酯(MTP),和异戊烯三磷酸或二甲基烯丙基三磷酸(ITP/DMATP),首次使用知识驱动的预测策略确定。我们进一步探索了这些新型代谢物的组织分布。总的来说,这项工作为所有MVA中间体开发了一种灵敏的定量方法,这将增强我们对这种途径在各种健康和疾病状况中的作用的理解。我们发现的新型代谢物需要对其生物合成和生物学功能进行进一步研究。
The mevalonate (MVA) pathway plays a crucial role in the occurrence and progression of various diseases, such as osteoporosis, breast cancer, and lung cancer, etc. However, determining all the MVA pathway intermediates is still challenging due to their high polarity, low concentration, chelation effect with metal compartments, and poor mass spectrometric response. In this study, we established a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method coupled with N2, N2, N4, N4-tetramethyl-6-(4-(piperazin-1-ylsulfonyl) phenyl)-1,3,5-triazine-2,4-diamine (Tmt-PP) labeling for the simultaneous analysis of all MVA intermediates in biospecimens. Chemical derivatization significantly improved the chromatographic retention, peak shape, and detection sensitivity of the analytes. Moreover, we employed a method named mass spectrum calculation to achieve the absolute quantification of the isomers, i.e., isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP). The established method was fully qualified and applied to explore the difference of these metabolites in cisplatin-resistant non-small cell lung cancer (NSCLC) cells. Additionally, several MVA intermediate analogs, including isopentenyl monophosphate or dimethylallyl monophosphate (IMP/DMAMP), geranyl monophosphate (GMP), 5-triphosphomevalonate (MTP), and isopentenyl triphosphate or dimethylallyl triphosphate (ITP/DMATP), were identified for the first time using a knowledge-driven prediction strategy. We further explored the tissue distribution of these novel metabolites. Overall, this work developed a sensitive quantification method for all MVA intermediates, which will enhance our understanding of the role of this pathway in various health and disease conditions. The novel metabolites we discovered warrant further investigations into their biosynthesis and biological functions.