PDF(Pubmed)
Abstract：
Metabolite extraction is the critical first-step in metabolomics experiments, where it is generally regarded to inactivate and remove proteins. Here, arising from efforts to improve extraction conditions for polar metabolomics, we discover a proteomic landscape of over 1000 proteins within metabolite extracts. This is a ubiquitous feature across several common extraction and sample types. By combining post-resuspension stable isotope addition and enzyme inhibitors, we demonstrate in-extract metabolite interconversions due to residual transaminase activity. We extend these findings with untargeted metabolomics where we observe extensive protein-mediated metabolite changes, including in-extract formation of glutamate dipeptide and depletion of total glutathione. Finally, we present a simple extraction workflow that integrates 3 kDa filtration for protein removal as a superior method for polar metabolomics. In this work, we uncover a previously unrecognized, protein-mediated source of observer effects in metabolomics experiments with broad-reaching implications across all research fields using metabolomics and molecular metabolism.
摘要：
代谢物提取是代谢组学实验中关键的第一步,它通常被认为是失活和去除蛋白质。这里,由于努力改善极性代谢组学的提取条件，我们发现了代谢产物提取物中超过1000种蛋白质的蛋白质组景观。这是几种常见提取和样本类型中普遍存在的特征。通过结合重悬后稳定同位素添加和酶抑制剂，我们证明了由于残留的转氨酶活性，提取物中的代谢物相互转化。我们通过非靶向代谢组学扩展了这些发现，我们观察到广泛的蛋白质介导的代谢物变化，包括谷氨酸二肽的提取物形成和总谷胱甘肽的消耗。最后,我们提出了一个简单的提取工作流程，该工作流程集成了3kDa过滤以去除蛋白质，作为极性代谢组学的优越方法。在这项工作中,我们发现了一个以前无法识别的，在代谢组学实验中，蛋白质介导的观察者效应来源在使用代谢组学和分子代谢的所有研究领域具有广泛的意义。
