International Institute for Translational Chinese Medicine , Guangzhou University of Chinese Medicine , Guangzhou , Guangdong 510006 , People's Republic of China.
Guangdong Key Laboratory for Translational Cancer Research of Chinese Medicine , Guangzhou University of Chinese Medicine , Guangzhou , Guangdong 510006 , People's Republic of China.
Anal Chem. 2019 Nov 19;91(22):14458-14466. doi: 10.1021/acs.analchem.9b03121. Epub 2019 Oct 30.
LC-MS-based untargeted metabolomics have been proven to be an extremely promising technique to discover biomarkers and explore the mechanisms underlying diseases, which, however, relies heavily on sample pretreatment for metabolite extraction. In the present study, a systematic and pragmatic evaluation of eight protocols employing conventional metabolites extraction strategies, protein precipitation (PPT), and liquid-liquid extraction (LLE), with and without proteinase K (PK) incubation, was performed simultaneously, using human plasma and a mixture of 39 endogenous metabolite standards. These protocols were as follows: (1) PPT with methanol, (2) PPT with acetonitrile, (3) PPT with 2-propanol, (4) two-step LLE of CHCl-MeOH, followed by MeOH-HO, (5) PK incubation combining two-step LLE of CHCl-MeOH followed by MeOH-HO, (6) two-step LLE of CHCl-MeOH, followed by MeOH-HO, (7) PK incubation combining two-step LLE of CHCl-MeOH, followed by MeOH-HO, (8) PK incubation combining MeOH-EtOH PPT. The results suggested that two-step LLE produced broader metabolome coverage than protein precipitation, and the addition of proteinase K enhanced the extraction performance further. Taken together, PK incubation combining two-step LLE of CHCl-MeOH, followed by MeOH-HO, was determined to be the most suitable extraction method, because of its broad metabolome coverage, high reproducibility, and satisfactory recovery. Next, the developed optimal sample preparation method was applied successfully to profile the plasma metabolome of colorectal adenoma and uncover its potential mechanism for significant differential changes in linoleic acid and phospholipid metabolism.
基于 LC-MS 的非靶向代谢组学已被证明是发现生物标志物和探索疾病机制的极具前景的技术，然而，该技术严重依赖于用于代谢物提取的样品预处理。在本研究中，同时对八种采用常规代谢物提取策略（蛋白质沉淀（PPT）和液液萃取（LLE））的方案进行了系统和务实的评估，这些方案包括有无蛋白酶 K（PK）孵育，使用人血浆和 39 种内源性代谢物标准混合物。这些方案如下：（1）甲醇中的 PPT，（2）乙腈中的 PPT，（3）2-丙醇中的 PPT，（4）二氯甲烷-甲醇两步 LLE，随后是甲醇-水，（5）PK 孵育结合二氯甲烷-甲醇两步 LLE，随后是甲醇-水，（6）二氯甲烷-甲醇两步 LLE，随后是甲醇-水，（7）PK 孵育结合二氯甲烷-甲醇两步 LLE，随后是甲醇-水，（8）PK 孵育结合甲醇-乙醇 PPT。结果表明，两步 LLE 产生的代谢组覆盖范围比蛋白质沉淀更广泛，而添加蛋白酶 K 进一步提高了提取性能。总的来说，PK 孵育结合二氯甲烷-甲醇两步 LLE，随后是甲醇-水，被确定为最适合的提取方法，因为它具有广泛的代谢组覆盖范围、高重现性和令人满意的回收率。接下来，成功地将开发的最佳样品制备方法应用于结直肠腺瘤的血浆代谢组学分析，揭示了其在亚油酸和磷脂代谢方面显著差异变化的潜在机制。
