Development of a high-sensitivity 13C NMR probe for metabolomics

项目摘要 代谢物对遗传和环境因素很敏感，因此是疾病的良好指标。 或表型。代谢组学的总体目标是测量与一种 特定的疾病、治疗方法、基因等。与其他组学相结合，代谢组学正在成为 在系统生物学研究、精密医学、食品和农业工业以及基于细胞的研究中不可或缺 制药公司。代谢组学的主要困难是可靠和可重复性的鉴定和 代谢物的量化。核磁共振和LC-MS等分析技术可以提供数百到数十种 来自代谢组学样本的数千个峰，但有效地和自信地量化这些峰 将它们分配给真正的代谢物仍然是一个巨大的挑战。核磁共振的标准方法 代谢组学是为了检测1H，因为它既丰富又敏感。1H核磁共振的问题是 峰往往是重叠的，这使得可靠的识别和量化变得困难。我们开发了新的 天然丰度和同位素条件下利用核磁共振检测13C的代谢组学方法 浓缩，以利用由于13C和13C的大光谱色散而减少峰重叠的优点 更可靠的化学转移到代谢物的数据库匹配。~(13)C核磁共振的主要局限性 代谢组学是敏感的。我们建议开发一种5毫米13C优化的800 MHz核磁共振探头，由 高温超导体(HTS)将至少提高代谢组学样品的灵敏度 比目前可用的高出3倍。这一灵敏度的提高将使测量时间缩短 至少是9倍的倍数，否则我们就可以在3倍的低浓度下检测到代谢物。这些改进 将与使用2个核磁共振接收器的新捕获方法相结合，并将在新的800上实施 用于增强灵敏度和吞吐量的MHz核磁共振光谱仪。基于13C信号比的目标值 噪声为9000：1对于ASTM标准，我们预计能够完全量化和识别高达130左右 代谢产物在类似人血清的生物流体中大约2小时。我们还在开发分馏的方法 并使用高效液相和固相萃取(SPE)浓缩样品，这项技术将使我们能够 也要测量相同样品的质谱学数据。我们应该能描述出300多个 SPE浓度为5倍的代谢物，或SPE为10倍的450种代谢物。这个项目会有很大的改进 代谢组学的重复性、可靠性和生物信息量。我们会向市民发放 通过商业化或向感兴趣的调查人员提供图纸来开发技术。 目标1)开发18.8T 5 mm 13C优化高温超导探头，将安装在Bruker Avance III HD上 乔治亚大学复合碳水化合物研究中心(CCRC)的核磁共振波谱仪。 目的2)开发新的代谢组学应用，使用2个带有定量13C 1D和 同时进行1H-2D核磁共振实验。LC-SPE将允许浓缩并与MS偶联。

Project Summary Metabolites are sensitive to genetic and environmental factors, and as a result are good indicators of disease or phenotype. The overall goal of metabolomics is the measurement of all metabolites associated with a specific disease, treatment, genotype, etc. Combined with other `omics, metabolomics is becoming indispensable in systems biology studies, precision medicine, food and agricultural industry, and cell-based pharmaceuticals. The major difficulty in metabolomics is the reliable and reproducible identification and quantification of metabolites. Analytical technologies such as NMR and LC-MS can provide hundreds to tens of thousands of peaks from metabolomics samples, but efficiently quantifying these peaks and confidently assigning them to real metabolites remains a significant challenge. The standard approach to NMR metabolomics is to detect 1H, because it is both abundant and sensitive. The problem with 1H NMR is that peaks are often overlapped, making reliable identification and quantification difficult. We have developed new approaches to metabolomics using 13C detection by NMR, both at natural abundance and with isotopic enrichment, to exploit the advantages of reduced peak overlap due to large spectral dispersion of 13C and more robust database matching of chemical shifts to metabolites. The primary limitation of 13C-based NMR metabolomics is sensitivity. We propose to develop a 5-mm 13C-optimized 800 MHz NMR probe made from high-temperature superconductors (HTS) that will improve the sensitivity for metabolomics samples by at least a factor of 3 beyond what is currently available. This sensitivity increase will reduce measurement times by at least a factor of 9x or it will allow us to detect metabolites at 3-fold lower concentrations. These improvements will be coupled with new acquisition methods using 2 NMR receivers and will be implemented on a new 800 MHz NMR spectrometer for enhanced sensitivity and throughput. Based on the target value for 13C signal-to- noise of 9000:1 for the ASTM standard, we expect to be able to fully quantify and identify up to around 130 metabolites in a biofluid like human serum in about 2 hours. We are also developing methods to fractionate and concentrate samples using HPLC and solid phase extraction (SPE), and this technology will allow us to also measure mass spectrometry data on the same samples. We should be able to characterize over 300 metabolites with a 5x SPE concentration, or 450 metabolites with a 10x SPE. This project will greatly improve the reproducibility, reliability, and biological information content of metabolomics. We will disseminate the technology through commercialization or by making the drawings available to interested investigators. Aim 1) Develop an 18.8 T 5-mm 13C-optimized HTS probe that will be installed on a Bruker Avance III HD NMR spectrometer in the Complex Carbohydrate Research Center (CCRC) at the University of Georgia. Aim 2) Develop new metabolomics applications using 2 receivers with quantitative 13C 1D and simultaneous 1H 2D NMR experiments. LC-SPE will allow concentration and coupling with MS.