Enhancing coverage in LC-MS-based untargeted metabolomics by a new sample preparation procedure using mixed-mode solid-phase extraction and two derivatizations
Enhancing coverage in LC-MS-based untargeted metabolomics by a new sample preparation procedure using mixed-mode solid-phase extraction and two derivatizations
复制标题
通过使用混合模式固相萃取和两次衍生化的新样品制备程序，增强基于 LC-MS 的非靶向代谢组学的覆盖范围
DOI：
10.1007/s00216-019-02010-x
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发表时间：
2019
影响因子：
4.3
通讯作者：
Lu Hongmei
中科院分区：
文献类型：
--
作者：
Wu Qian;Xu Yamei;Ji Hongchao;Wang Yang;Zhang Zhimin;Lu Hongmei
扩大基于液相色谱（LC）-电喷雾电离（ESI）质谱（MS）的非靶向代谢组学分析的代谢组覆盖范围是一个挑战。有限的覆盖范围归因于ESI-MS中羟基和羧基的弱信号以及LC分离具有广泛极性的代谢物的能力有限。本文提出了一种新的样品制备方法来解决这些问题。混合模式（反相和阴离子交换）的固相萃取吸附剂被用来分离代谢物成亲水性胺，疏水性胺/醇，和有机酸基团。然后，醇和羧酸在分离的基团与吡啶标记使用两个衍生化系统的信号增强。最后，亲水性胺用亲水相互作用LC柱通过LC-MS分析，两个疏水化合物基团用C18柱通过LC-MS分析。对标准样品的测定结果表明，新方法的检出限比经典的溶剂萃取-蛋白质沉淀法低3.3 ~ 70倍（5种氨基酸）和65-1141倍（5种脂肪酸）。该方法对胆固醇的检出限为125 ng mL-1，与经典方法相比，胆固醇的检出限为10 μg mL-1。在精浆样品中，用这种新方法比传统的溶剂提取-蛋白沉淀法在正离子模式ESI中多鉴定了110种代谢物（新方法与传统方法，通过比较MS/MS谱与标准品谱鉴定了65种与22种，通过在已发表的数据库中搜索MS谱鉴定了203种与136种）。新方法仅鉴定了53种羧酸类和21种醇类，比经典方法鉴定了更多的氨基酸、核苷等亲水性胺类代谢产物。最后，在应用于男性不育症的研究中，新方法发现了更多的潜在生物标志物（46个潜在生物标志物），而不是经典方法（19个潜在生物标志物）和以前报道的方法（10-30个潜在生物标志物）。因此，证明了这种新的样品制备方法扩大了基于LC-MS的非靶向代谢组学方法的检测范围，并在生物研究中具有应用潜力。
It is a challenge to expand the metabolome coverage of liquid chromatography (LC)–electrospray ionization (ESI) mass spectrometry (MS) based untargeted metabolomics analysis. The limited coverage is attributed to the weak signal of hydroxyl and carboxyl groups in ESI-MS and the limited capacity of LC separation for metabolites with a wide range of polarities. Here a new sample preparation procedure is proposed to solve these problems. Mixed-mode (reversed-phase and anion-exchange) solid-phase extraction sorbents were used to separate metabolites into hydrophilic amine, hydrophobic amine/alcohol, and organic acid groups. Then, alcohols and carboxylic acids in separated groups were tagged with pyridine with use of two derivatization systems for signal enhancement. Finally, hydrophilic amines were analyzed by LC–MS with a hydrophilic interaction LC column, and the two hydrophobic compound groups were analyzed by LC–MS with a C18column. From the results for standard samples, the detection limits of the new method are lower than those of the classic solvent extraction–protein precipitation method by 3.3–70 times for five amino acids and by 65–1141 times for five fatty acids. Moreover, the detection limit of this new method is 125 ng mL-1for cholesterol, which has no signal with the classic method even at 10 μg mL-1. In seminal plasma samples, 110 more metabolites were identified by this new method than by the traditional solvent extraction–protein precipitation method in positive-mode ESI (new method vs traditional method, 65 vs 22 identified by comparing MS/MS spectra with those of standards, 203 vs 136 identified by searching MS spectra in a published database). Among them, 53 carboxylic acids and 21 alcohols were identified only by the new method, and more hydrophilic amine metabolites, such as amino acids and nucleosides, were identified by the new method than by the classic method. Finally, in application to the study of male infertility, more potential biomarkers of oligoasthenoteratospermic infertility were found with the new method (46 potential biomarkers) than with the classic method (19 potential biomarkers) and previously reported methods (10–30 potential biomarkers). Thus, it is demonstrated that this new sample preparation method expands the detection coverage of LC–MS-based untargeted metabolomics methods and has application potential in biological research.