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代谢相关的所有代谢物。 具体疾病、治疗、基因型等。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 大药厂代谢组学的主要困难是可靠的和可重复的鉴定， 代谢物的定量。分析技术如NMR和LC-MS可以提供数百至数十个 来自代谢组学样品的数千个峰，但有效地量化这些峰， 将它们分配给真实的代谢物仍然是一个重大的挑战。NMR的标准方法 代谢组学是检测1H，因为它既丰富又敏感。1H NMR的问题在于， 峰经常重叠，使得难以进行可靠的鉴定和定量。我们开发了新 使用NMR进行13 C检测的代谢组学方法，包括天然丰度和同位素 富集，以利用由于13 C的大光谱色散而减少的峰重叠的优点， 化学位移与代谢物的数据库匹配更加稳健。13 C核磁共振的主要局限性 代谢组学就是敏感性。我们建议开发一种5 mm 13 C优化的800 MHz NMR探头， 高温超导体（HTS）将提高代谢组学样本的灵敏度，至少 是目前可用的三倍。这种灵敏度的提高将减少测量时间 至少是9倍，否则它将允许我们检测浓度低3倍的代谢物。这些改进 将与使用2个NMR接收器的新采集方法相结合，并将在新的800 MHz核磁共振光谱仪，用于增强灵敏度和通量。根据13 C信号的目标值， 对于ASTM标准的9000：1的噪声，我们希望能够完全量化和识别高达约130 代谢物在生物流体如人血清中的浓度。我们也在开发压裂方法 并使用HPLC和固相萃取（SPE）浓缩样品，这项技术将使我们能够 还测量相同样品的质谱数据。我们应该能找出300多个 5x SPE浓度的代谢物，或10 x SPE浓度的450种代谢物。该项目将大大改善 代谢组学的可重复性、可靠性和生物信息含量。我们将传播 通过商业化或向感兴趣的研究人员提供图纸来获得技术。 目标1）开发一种18.8 T 5 mm 13 C优化的HTS探头，将安装在Bruker Avance III HD上 格鲁吉亚大学复杂碳水化合物研究中心（CCRC）的NMR光谱仪。 目的2）使用2个定量13 C 1D和13 C 1D的接收器开发新的代谢组学应用。 同时进行1H 2D NMR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.