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首页> 外文期刊>Journal of chromatography, A: Including electrophoresis and other separation methods >LC-MS/MS analysis of the central energy and carbon metabolites in biological samples following derivatization by dimethylaminophenacyl bromide
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LC-MS/MS analysis of the central energy and carbon metabolites in biological samples following derivatization by dimethylaminophenacyl bromide

机译：通过二甲基氨基苯基苯甲酰基衍生化后生物样品中的中央能量和碳代谢物的LC-MS / MS分析

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摘要

Recent advances in metabolomics have enabled larger proportions of the human metabolome to be analyzed quantitatively. However, this usually requires the use of several chromatographic methods coupled to mass spectrometry to cover the wide range of polarity, acidity/basicity and concentration of metabolites. Chemical derivatization allows in principle a wide coverage in a single method, as it affects both the separation and the detection of metabolites: it increases retention, stabilizes the analytes and improves the sensitivity of the analytes. The majority of quantitative derivatization techniques for LC-MS in metabolomics react with amines, phenols and thiols; however, there are unfortunately very few methods that can target carboxylic acids at the same time, which contribute to a large proportion of the human metabolome. Here, we describe a derivatization technique which simultaneously labels carboxylic acids, thiols and amines using the reagent dimethylaminophenacyl bromide (DmPABr). We further improve the quantitation by employing isotope-coded derivatization (lCD), which uses internal standards derivatized with an isotopically-labelled reagent (DmPABr-D-6). We demonstrate the ability to measure and quantify 64 central carbon and energy-related metabolites including amino acids, N-acetylated amino acids, metabolites from the TCA cycle and pyruvate metabolism, acylcarnitines and medium-/long-chain fatty acids. To demonstrate the applicability of the analytical approach, we analyzed urine and SUIT-2 cells utilizing a 15-minute single UPLC-MS/MS method in positive ionization mode. SUIT-2 cells exposed to rotenone showed definitive changes in 28 out of the 64 metabolites, including metabolites from all 7 classes mentioned. By realizing the full potential of DmPABr to derivatize and quantify amines and thiols in addition to carboxylic acids, we extended the coverage of the metabolome, producing a strong platform that can be further applied to a variety of biological studies. (C) 2019 The Authors. Published by Elsevier B.V.
机译：最近代谢组学的进展使得量化的人类代谢物的更大比例是定量分析的。然而，这通常需要使用偶联的几种色谱方法，以覆盖各种极性，酸度/碱度和代谢物的浓度。化学衍生化允许原则上以单一方法宽覆盖，因为它会影响代谢物的分离和检测：它会增加保留，稳定分析物并提高分析物的敏感性。在代谢组科中LC-MS的大多数定量衍生化技术与胺，酚和硫醇反应;然而，遗憾的是，在靶向羧酸同时靶向羧酸的方法很少，这有助于较大比例的人代谢物。这里，我们描述了一种衍生化技术，其使用试剂二甲基氨基苯基酰基溴（DMPABR）同时标记羧酸，硫醇和胺。我们通过采用同位素编码的衍生化（LCD）进一步提高了定量，该衍生化（LCD）使用用同位素标记的试剂（DMPABR-D-6）衍生的内标。我们证明了测量和量化64个中央碳和能量相关代谢物，包括氨基酸，N-乙酰化氨基酸，来自TCA循环和丙酮酸代谢，酰基碱和中/长链脂肪酸的代谢物。为了证明分析方法的适用性，我们在正电离模式下分析了利用15分钟的单级UPLC-MS / MS方法的尿液和适用性。暴露于旋转晶酮的西装2细胞显示出64种代谢物中的28中的明确变化，包括来自所有7个类别的代谢物。通过实现DMPABR的全部潜力除羧酸之外，我们还延长了代谢物的覆盖率，产生了可以进一步应用于各种生物学研究的强平台。 （c）2019年作者。由elsevier b.v出版。

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