Department of Biopharmaceutics and Pharmacodynamics, Medical University of Gdańsk, Gdańsk, Poland.
Department of Biology and Pharmaceutical Botany, Medical University of Gdańsk, Gdańsk, Poland.
Sci Rep. 2018 Jun 22;8(1):9541. doi: 10.1038/s41598-018-27983-0.
Plasma untargeted metabolomics is a common method for evaluation of the mechanisms underlying human pathologies and identification of novel biomarkers. The plasma proteins provide the environment for transport of hydrophobic metabolites. The current sample preparation protocol relies on the immediate precipitation of proteins and thus leads to co-precipitation of a significant fraction of hydrophobic metabolites. Here we present a new simple procedure that overcomes the co-precipitation problem and improves metabolome coverage. Introducing an additional step preceding the protein precipitation, namely limited digestion with proteinase K, allows release of associated metabolites through the relaxation of the native proteins tertiary structure. The modified protocol allows clear detection of hydrophobic metabolites including fatty acids and phospholipids. Considering the potential involvement of the hydrophobic metabolites in human cardiovascular and cancer diseases, the method may constitute a novel approach in plasma untargeted metabolomics.
血浆非靶向代谢组学是评估人类病理机制和鉴定新型生物标志物的常用方法。血浆蛋白为疏水性代谢物的转运提供了环境。目前的样品制备方案依赖于蛋白质的即时沉淀，因此导致大量疏水性代谢物的共沉淀。在这里，我们提出了一种新的简单方法，该方法克服了共沉淀问题并提高了代谢组学的覆盖范围。在蛋白质沉淀之前引入一个额外的步骤，即使用蛋白酶 K 进行有限的消化，通过放松天然蛋白质的三级结构，使相关代谢物释放出来。该改进后的方案可清晰检测到包括脂肪酸和磷脂在内的疏水性代谢物。考虑到疏水性代谢物可能参与人类心血管和癌症疾病，该方法可能构成血浆非靶向代谢组学的新方法。
