Expanding the limits of human blood metabolite quantitation using NMR spectroscopy.
Expanding the limits of human blood metabolite quantitation using NMR spectroscopy.
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DOI：
10.1021/ac503651e
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发表时间：
2015-01-06
影响因子：
7.4
通讯作者：
Raftery, Daniel
中科院分区：
文献类型：
--
作者：
Gowda, G. A. Nagana;Gowda, Yashas N.;Raftery, Daniel
代谢组学目前面临的挑战是许多代谢物的可靠定量。有限的分辨率和灵敏度以及与未知代谢物鉴定相关的挑战限制了血液代谢物的数量和定量准确性。为了缓解基于NMR的代谢组学中的这一瓶颈，对合并的人血清进行了研究，结合了800 MHz的一系列1D/2D NMR实验、数据库搜索和真实化合物的加标，从而鉴定了67种血液代谢物。与以前作为人血清代谢组数据库的一部分报道的那些相比，其中许多（101/3）是新的。此外，考虑到NMR的高重现性和定量性质以及NMR化学位移对改变的样品条件的敏感性，本文提供了实验方案和全面的峰注释，作为常规应用中新血液代谢物池的鉴定和定量指南。此外，集中于使用有机溶剂定量评价的研究揭示了使用乙腈进行蛋白质沉淀的令人惊讶的差性能。在相同的溶剂-血清比2：1（v/v）下，与甲醇沉淀相比，三分之一的检测到的代谢物衰减了10-67%。当乙腈与血清的比例增加至4：1（v/v）时，近2/3的代谢产物进一步减毒，最多可达65%。这些结果，结合新建立的身份为许多未知的代谢产物的NMR光谱，提供了新的途径，为人类血清/血浆为基础的代谢组学。此外，使用简单且高度可重复的分析方法（如NMR）定量评价代表多种类别（包括氨基酸、有机酸、碳水化合物和杂环化合物）的近70种血液代谢物的能力可能会指导使用质谱法进行分析的样品评价。
A current challenge in metabolomics is the reliable quantitation of many metabolites. Limited resolution and sensitivity combined with the challenges associated with unknown metabolite identification have restricted both the number and the quantitative accuracy of blood metabolites. Focused on alleviating this bottleneck in NMR-based metabolomics, investigations of pooled human serum combining an array of 1D/2D NMR experiments at 800 MHz, database searches, and spiking with authentic compounds enabled the identification of 67 blood metabolites. Many of these (∼1/3) are new compared with those reported previously as a part of the Human Serum Metabolome Database. In addition, considering both the high reproducibility and quantitative nature of NMR as well as the sensitivity of NMR chemical shifts to altered sample conditions, experimental protocols and comprehensive peak annotations are provided here as a guide for identification and quantitation of the new pool of blood metabolites for routine applications. Further, investigations focused on the evaluation of quantitation using organic solvents revealed a surprisingly poor performance for protein precipitation using acetonitrile. One-third of the detected metabolites were attenuated by 10–67% compared with methanol precipitation at the same solvent-to-serum ratio of 2:1 (v/v). Nearly 2/3 of the metabolites were further attenuated by up to 65% upon increasing the acetonitrile-to-serum ratio to 4:1 (v/v). These results, combined with the newly established identity for many unknown metabolites in the NMR spectrum, offer new avenues for human serum/plasma-based metabolomics. Further, the ability to quantitatively evaluate nearly 70 blood metabolites that represent numerous classes, including amino acids, organic acids, carbohydrates, and heterocyclic compounds, using a simple and highly reproducible analytical method such as NMR may potentially guide the evaluation of samples for analysis using mass spectrometry.
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