Quantitating metabolites in protein precipitated serum using NMR spectroscopy.
Quantitating metabolites in protein precipitated serum using NMR spectroscopy.
复制标题
使用NMR光谱法定量蛋白质沉淀血清中的代谢产物。
DOI：
10.1021/ac5005103
复制
发表时间：
2014-06-03
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;Raftery, Daniel
基于定量NMR的代谢物谱分析受到完整血浆/血清中丰富蛋白质的有害影响的挑战，这强调了对替代方法的需求。因此，使用低分子量截留过滤器通过超滤去除蛋白质代表了重要的步骤。然而，蛋白质沉淀，蛋白质去除的替代和简单的方法，缺乏详细的定量评估用于基于NMR的代谢组学。在这项研究中，我们已经全面评估了蛋白质沉淀的性能，使用甲醇，乙腈，高氯酸，三氯乙酸和超滤方法，使用1D和2D NMR，基于44人血液代谢物的鉴定和绝对定量，包括一些首次在人血清的NMR光谱。我们还研究了使用“智能同位素标签”，15 N-胆碱进一步提高分辨率，这导致了一些额外的代谢物的检测。蛋白沉淀和超滤血清的1H NMR检测到所有44种代谢物，重现性相当（平均CV，沉淀为3.7%;过滤为3.6%）。然而，超滤血清中近一半的定量代谢物显示出10-74%的低浓度;具体而言，色氨酸、苯甲酸盐和2-氧代异己酸盐显示出比蛋白沉淀血清低得多的浓度。这些结果表明，蛋白质沉淀使用甲醇提供了一个可靠的方法，为常规的NMR为基础的代谢组学的人血清/血浆，应被视为一种替代超滤。重要的是，蛋白质沉淀，这是常用的质谱法（MS），承诺的途径，直接比较和相关的代谢物数据从两个分析平台，以利用其在血液代谢组学的综合实力。
Quantitative NMR-based metabolite profiling is challenged by the deleterious effects of abundant proteins in the intact blood plasma/serum, which underscores the need for alternative approaches. Protein removal by ultrafiltration using low molecular weight cutoff filters thus represents an important step. However, protein precipitation, an alternative and simple approach for protein removal, lacks detailed quantitative assessment for use in NMR based metabolomics. In this study, we have comprehensively evaluated the performance of protein precipitation using methanol, acetonitrile, perchloric acid, and trichloroacetic acid and ultrafiltration approaches using 1D and 2D NMR, based on the identification and absolute quantitation of 44 human blood metabolites, including a few identified for the first time in the NMR spectra of human serum. We also investigated the use of a “smart isotope tag,” 15N-cholamine for further resolution enhancement, which resulted in the detection of a number of additional metabolites. 1H NMR of both protein precipitated and ultrafiltered serum detected all 44 metabolites with comparable reproducibility (average CV, 3.7% for precipitation; 3.6% for filtration). However, nearly half of the quantified metabolites in ultrafiltered serum exhibited 10–74% lower concentrations; specifically, tryptophan, benzoate, and 2-oxoisocaproate showed much lower concentrations compared to protein precipitated serum. These results indicate that protein precipitation using methanol offers a reliable approach for routine NMR-based metabolomics of human blood serum/plasma and should be considered as an alternative to ultrafiltration. Importantly, protein precipitation, which is commonly used by mass spectrometry (MS), promises avenues for direct comparison and correlation of metabolite data obtained from the two analytical platforms to exploit their combined strength in the metabolomics of blood.
登录
查看更多内容
影响因子：
7.4
作者：
Bruce, Stephen J.;Tavazzi, Isabelle;Guy, Philippe A.
通讯作者：
Guy, Philippe A.
影响因子：
7.4
作者：
Jiye, A;Trygg, J;Moritz, T
通讯作者：
Moritz, T
影响因子：
37.8
作者：
Shah SH;Kraus WE;Newgard CB
通讯作者：
Newgard CB
影响因子：
2.7
作者：
Mercier, Pascal;Lewis, Michael J.;Wishart, David S.
通讯作者：
Wishart, David S.
DOI：
10.1016/s0304-4165(98)00154-8
发表时间：
1999-01-04
影响因子：
3
作者：
Chatham, JC;Forder, JR
通讯作者：
Forder, JR