Kacerova Tereza, Pires Elisabete, Walsby-Tickle John, Probert Fay, McCullagh James S O
Chemistry Research Laboratory, Department of Chemistry, University of Oxford, Oxford, OX1 3TA, UK.
Chemistry Research Laboratory, Department of Chemistry, University of Oxford, Oxford, OX1 3TA, UK.
Anal Chim Acta. 2025 Jun 22;1356:343979. doi: 10.1016/j.aca.2025.343979. Epub 2025 Apr 4.
Mass spectrometry (MS) and nuclear magnetic resonance (NMR) have emerged as pivotal tools in biofluid metabolomics, facilitating investigation of disease mechanisms and biomarker discovery. Despite complementary capabilities, these techniques are rarely combined, although their integration is often beneficial. Typically, different sample preparation approaches are used, and compatibility challenges potentially arise due to the requirement for deuterated buffered solvents in NMR but not MS techniques. Additionally, MS-based approaches necessitate protein removal from samples whilst in NMR proteins can be potentially useful biomarkers. In this study, we developed a blood serum preparation protocol enabling sequential NMR and multi-LC-MS untargeted metabolomics analysis using a single serum aliquot in a research discovery setting.
We analysed human serum samples using various untargeted NMR and multi-LC-MS platforms to assess the impact of deuterated solvents and buffers on detected compound-features. Employing multiple LC-MS profiling approaches, we observed no evidence of deuterium incorporation into metabolites following sample preparation with deuterated solvents. Furthermore, we demonstrated that buffers used in NMR were well tolerated by LC-MS. Protein removal, involving both solvent precipitation and molecular weight cut-off (MWCO) filtration, was identified as a primary factor influencing metabolite abundance. Our findings led to the development and validation of a serum sample preparation protocol enabling a combined NMR and multi-LC-MS analysis.
Using a single clinical serum aliquot for simultaneous untargeted profiling via NMR and multi-LC-MS represents a highly efficient alternative to current methods. This approach reduces sample volume requirements and substantially expands the potential for broader metabolome coverage. Our study offers comprehensive insights into the impact of sample preparation on complex metabolic biofluid profiles, highlighting the compatibility and complementarity of LC-MS and NMR in metabolomics research.
质谱（MS）和核磁共振（NMR）已成为生物流体代谢组学中的关键工具，有助于疾病机制研究和生物标志物发现。尽管这些技术具有互补性，但它们很少结合使用，尽管它们的整合通常是有益的。通常，使用不同的样品制备方法，并且由于NMR技术需要氘代缓冲溶剂而MS技术不需要，可能会出现兼容性挑战。此外，基于MS的方法需要从样品中去除蛋白质，而在NMR中蛋白质可能是潜在有用的生物标志物。在本研究中，我们开发了一种血清制备方案，能够在研究发现环境中使用单个血清等分试样进行顺序NMR和多LC-MS非靶向代谢组学分析。
我们使用各种非靶向NMR和多LC-MS平台分析了人类血清样品，以评估氘代溶剂和缓冲液对检测到的化合物特征的影响。采用多种LC-MS分析方法，我们观察到在用氘代溶剂制备样品后，没有证据表明氘掺入代谢物中。此外，我们证明了NMR中使用的缓冲液对LC-MS具有良好的耐受性。涉及溶剂沉淀和分子量截留（MWCO）过滤的蛋白质去除被确定为影响代谢物丰度的主要因素。我们的研究结果导致了一种血清样品制备方案的开发和验证，该方案能够进行联合NMR和多LC-MS分析。
使用单个临床血清等分试样通过NMR和多LC-MS同时进行非靶向分析是目前方法的一种高效替代方案。这种方法减少了样品体积需求，并大大扩展了更广泛代谢组覆盖的潜力。我们的研究提供了关于样品制备对复杂代谢生物流体谱影响的全面见解，突出了LC-MS和NMR在代谢组学研究中的兼容性和互补性。
