García-Cañaveras Juan Carlos, López Silvia, Castell José Vicente, Donato M Teresa, Lahoz Agustín
Unidad de Hepatología Experimental, Unidad Analítica, Instituto de Investigación Sanitaria, Fundación Hospital La Fe, Av. Fernando Abril Martorell 106, Valencia, 46026, Spain.
Departamento de Bioquímica y Biología Molecular, Facultad de Medicina y Odontología, Universidad de Valencia, Av. Blasco Ibáñez 15, 46010, Valencia, Spain.
Anal Bioanal Chem. 2016 Feb;408(4):1217-30. doi: 10.1007/s00216-015-9227-8. Epub 2016 Jan 14.
MS-based metabolite profiling of adherent mammalian cells comprises several challenging steps such as metabolism quenching, cell detachment, cell disruption, metabolome extraction, and metabolite measurement. In LC-MS, the final metabolome coverage is strongly determined by the separation technique and the MS conditions used. Human liver-derived cell line HepG2 was chosen as adherent mammalian cell model to evaluate the performance of several commonly used procedures in both sample processing and LC-MS analysis. In a first phase, metabolite extraction and sample analysis were optimized in a combined manner. To this end, the extraction abilities of five different solvents (or combinations) were assessed by comparing the number and the levels of the metabolites comprised in each extract. Three different chromatographic methods were selected for metabolites separation. A HILIC-based method which was set to specifically separate polar metabolites and two RP-based methods focused on lipidome and wide-ranging metabolite detection, respectively. With regard to metabolite measurement, a Q-ToF instrument operating in both ESI (+) and ESI (-) was used for unbiased extract analysis. Once metabolite extraction and analysis conditions were set up, the influence of cell harvesting on metabolome coverage was also evaluated. Therefore, different protocols for cell detachment (trypsinization or scraping) and metabolism quenching were compared. This study confirmed the inconvenience of trypsinization as a harvesting technique, and the importance of using complementary extraction solvents to extend metabolome coverage, minimizing interferences and maximizing detection, thanks to the use of dedicated analytical conditions through the combination of HILIC and RP separations. The proposed workflow allowed the detection of over 300 identified metabolites from highly polar compounds to a wide range of lipids.
基于质谱的贴壁哺乳动物细胞代谢物谱分析包含几个具有挑战性的步骤，如代谢淬灭、细胞脱离、细胞破碎、代谢组提取和代谢物测量。在液相色谱-质谱联用（LC-MS）中，最终的代谢组覆盖范围很大程度上取决于所使用的分离技术和质谱条件。选择人肝源性细胞系HepG2作为贴壁哺乳动物细胞模型，以评估几种常用方法在样品处理和LC-MS分析中的性能。在第一阶段，以组合方式优化代谢物提取和样品分析。为此，通过比较每种提取物中所含代谢物的数量和水平，评估了五种不同溶剂（或组合）的提取能力。选择了三种不同的色谱方法进行代谢物分离。一种基于亲水相互作用色谱（HILIC）的方法专门用于分离极性代谢物，另外两种基于反相色谱（RP）的方法分别侧重于脂质组和广泛代谢物的检测。关于代谢物测量，使用在电喷雾电离（ESI）（+）和ESI（-）模式下运行的四极杆飞行时间（Q-ToF）仪器对提取物进行无偏分析。一旦建立了代谢物提取和分析条件，还评估了细胞收获对代谢组覆盖范围的影响。因此，比较了不同的细胞脱离方案（胰蛋白酶消化或刮擦）和代谢淬灭方法。这项研究证实了胰蛋白酶消化作为一种收获技术的不便之处，以及使用互补提取溶剂以扩大代谢组覆盖范围、最小化干扰并最大化检测的重要性，这得益于通过结合HILIC和RP分离使用专门的分析条件。所提出的工作流程能够检测从高极性化合物到多种脂质的300多种已鉴定代谢物。
