Zhang Lun, Zheng Jiamin, Johnson Mathew, Mandal Rupasri, Cruz Meryl, Martínez-Huélamo Miriam, Andres-Lacueva Cristina, Wishart David S
Department of Biological Sciences, University of Alberta, Edmonton, AB T6G 2E8, Canada.
Biomarkers and Nutrimetabolomics Laboratory, Department of Nutrition, Food Sciences and Gastronomy, Nutrition and Food Safety Research Institute (INSA-UB), Faculty of Pharmacy and Food Sciences, University of Barcelona, 08028 Barcelona, Spain.
Metabolites. 2024 Nov 14;14(11):622. doi: 10.3390/metabo14110622.
Targeted metabolomics is often criticized for the limited metabolite coverage that it offers. Indeed, most targeted assays developed or used by researchers measure fewer than 200 metabolites. In an effort to both expand the coverage and improve the accuracy of metabolite quantification in targeted metabolomics, we decided to develop a comprehensive liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay that could quantitatively measure more than 700 metabolites in serum or plasma. The developed assay makes use of chemical derivatization followed by reverse phase LC-MS/MS and/or direct flow injection MS (DFI-MS) in both positive and negative ionization modes to separate metabolites. Multiple reaction monitoring (MRM), in combination with isotopic standards and multi-point calibration curves, is used to detect and absolutely quantify the targeted metabolites. The assay has been adapted to a 96-well plate format to enable automated, high-throughput sample analysis. The assay (called MEGA) is able to detect and quantify 721 metabolites in serum/plasma, covering 20 metabolite classes and many commonly used clinical biomarkers. The limits of detection were determined to range from 1.4 nM to 10 mM, recovery rates were from 80% to 120%, and quantitative precision was within 20%. LC-MS/MS metabolite concentrations of the NIST SRM1950 plasma standard were found to be within 15% of NMR quantified levels. The MEGA assay was further validated in a large dietary intervention study. The MEGA assay should make comprehensive quantitative metabolomics much more affordable, accessible, automatable, and applicable to large-scale clinical studies.
靶向代谢组学常常因其提供的代谢物覆盖范围有限而受到批评。确实，研究人员开发或使用的大多数靶向分析方法测量的代谢物不到200种。为了扩大靶向代谢组学中代谢物覆盖范围并提高代谢物定量的准确性，我们决定开发一种全面的液相色谱 - 串联质谱（LC-MS/MS）分析方法，该方法可以定量测量血清或血浆中的700多种代谢物。所开发的分析方法利用化学衍生化，然后在正离子和负离子模式下进行反相LC-MS/MS和/或直接流动注射质谱（DFI-MS）来分离代谢物。多反应监测（MRM）结合同位素标准和多点校准曲线，用于检测和绝对定量靶向代谢物。该分析方法已适用于96孔板形式，以实现自动化、高通量的样品分析。该分析方法（称为MEGA）能够检测和定量血清/血浆中的721种代谢物，涵盖20类代谢物和许多常用的临床生物标志物。检测限确定为1.4 nM至10 mM，回收率为80%至120%，定量精度在20%以内。发现NIST SRM1950血浆标准品的LC-MS/MS代谢物浓度在NMR定量水平的15%以内。MEGA分析方法在一项大型饮食干预研究中得到了进一步验证。MEGA分析方法应使全面的定量代谢组学更经济实惠、更易于获得、更可自动化，并且适用于大规模临床研究。
