了解蛋白质组和代谢组的相互作用有助于了解细胞调节和反应。为了从此类多组学分析中得出可靠的推论，我们引入并评估了从单个样本开始的蛋白质组和代谢组组合分析的工作流程。具体来说，我们将已建立的和单独优化的代谢组学和蛋白质组学分析方案（分别为 EtOH/MTBE 和 autoSP3）集成到统一的工作流程（称为 MTBE-SP3）中，并利用了代谢组学样品的蛋白质残留可以被分析的事实。用作蛋白质组分析的直接输入。我们特别评估了 MTBE-SP3 中蛋白质组分析的性能，并证明了蛋白质组图谱的等效性，而与先前的代谢物提取无关。此外，MTBE-SP3结合了EtOH/MTBE和autoSP3的优点，分别用于半自动代谢物提取和全自动蛋白质组样品制备，从而提高了大规模研究的标准化和可扩展性。我们证明，MTBE-SP3 可应用于各种生物基质（FFPE 组织、新鲜冷冻组织、血浆、血清和细胞），从而能够在各种临床环境中实施。为了证明适用性，我们将 MTBE-SP3 和 autoSP3 应用到肺腺癌队列中，显示肿瘤和非肿瘤相邻组织之间存在一致的蛋白质组变化，而与所使用的方法无关。与从相同样本获得的代谢组学数据的整合揭示了肿瘤组织中通过 OGDH、SDH 家族酶和 PKM 失调而出现的线粒体功能障碍。总之，MTBE-SP3 能够轻松可靠地并行测量从同一样品中获得的蛋白质和代谢物，受益于减少的样品变化和输入量。该工作流程特别适用于样本可用性有限的研究，并提供了增强代谢组学和蛋白质组学数据集集成的潜力。© 2024。作者。

Understanding the interplay of the proteome and the metabolome helps to understand cellular regulation and response. To enable robust inferences from such multi-omics analyses, we introduced and evaluated a workflow for combined proteome and metabolome analysis starting from a single sample. Specifically, we integrated established and individually optimized protocols for metabolomic and proteomic profiling (EtOH/MTBE and autoSP3, respectively) into a unified workflow (termed MTBE-SP3), and took advantage of the fact that the protein residue of the metabolomic sample can be used as a direct input for proteome analysis. We particularly evaluated the performance of proteome analysis in MTBE-SP3, and demonstrated equivalence of proteome profiles irrespective of prior metabolite extraction. In addition, MTBE-SP3 combines the advantages of EtOH/MTBE and autoSP3 for semi-automated metabolite extraction and fully automated proteome sample preparation, respectively, thus advancing standardization and scalability for large-scale studies. We showed that MTBE-SP3 can be applied to various biological matrices (FFPE tissue, fresh-frozen tissue, plasma, serum and cells) to enable implementation in a variety of clinical settings. To demonstrate applicability, we applied MTBE-SP3 and autoSP3 to a lung adenocarcinoma cohort showing consistent proteomic alterations between tumour and non-tumour adjacent tissue independent of the method used. Integration with metabolomic data obtained from the same samples revealed mitochondrial dysfunction in tumour tissue through deregulation of OGDH, SDH family enzymes and PKM. In summary, MTBE-SP3 enables the facile and reliable parallel measurement of proteins and metabolites obtained from the same sample, benefiting from reduced sample variation and input amount. This workflow is particularly applicable for studies with limited sample availability and offers the potential to enhance the integration of metabolomic and proteomic datasets.© 2024. The Author(s).