Berlin Institute of Health (BIH) @ Charité, BIH Metabolomics Platform, Anna-Louisa-Karsch-Str. 2, 10178, Berlin, Germany.
Core Unit Bioinformatics, Berlin Institute of Health (BIH) @ Charité, Chariteplatz 1, 10117, Berlin, Germany.
Sci Rep. 2022 May 13;12(1):7933. doi: 10.1038/s41598-022-11118-7.
The AbsoluteIDQ p400 HR kit is a commercial product for targeted metabolomics. While the kit has been validated for human plasma and serum, adherent cell lysates have not yet been evaluated. We have optimized the detection of polar and lipid metabolites in cell lysates using the kit to enable robust and repeatable analysis of the detected metabolites. Parameters optimized include total cell mass, loading volume and extraction solvent. We present a cell preparation and analytical method and report on the performance of the kit with regard to detectability of the targeted metabolites and their repeatability. The kit can be successfully used for a relative quantification analysis of cell lysates from adherent cells although validated only for human plasma and serum. Most metabolites are below the limit of the Biocrates' set quantification limits and we confirmed that this relative quantification can be used for further statistical analysis. Using this approach, up to 45% of the total metabolites in the kit can be detected with a reasonable analytical performance (lowest median RSD 9% and 13% for LC and FIA, respectively) dependent on the method used. We recommend using ethanol as the extraction solvent for cell lysates of osteosarcoma cell lines for the broadest metabolite coverage and 25 mg of cell mass with a loading volume of 20 µL per sample.
绝对 IDQ p400 HR 试剂盒是一种用于靶向代谢组学的商业产品。虽然该试剂盒已经过人类血浆和血清的验证，但尚未评估贴壁细胞裂解物。我们已经使用该试剂盒对细胞裂解物中的极性和脂质代谢物进行了优化检测，以实现对检测到的代谢物的稳健和可重复分析。优化的参数包括总细胞质量、加载体积和提取溶剂。我们提出了一种细胞制备和分析方法，并报告了试剂盒在靶向代谢物的检测能力及其重复性方面的性能。该试剂盒可以成功用于贴壁细胞裂解物的相对定量分析，尽管仅针对人血浆和血清进行了验证。大多数代谢物低于 Biocrates 设定的定量限，我们证实这种相对定量可用于进一步的统计分析。使用这种方法，取决于所使用的方法，试剂盒中多达 45%的总代谢物可以以合理的分析性能（最低中位数 RSD 分别为 LC 和 FIA 的 9%和 13%）进行检测。我们建议使用乙醇作为骨肉瘤细胞系细胞裂解物的提取溶剂，以获得最广泛的代谢物覆盖范围，每个样本的细胞质量为 25mg，加载体积为 20µL。
