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

项目概要 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某个物质相关的所有代谢物。 特定疾病、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、精准医学、食品和农业以及细胞基础研究中不可或缺 药品。代谢组学的主要困难是可靠且可重复的鉴定和 代谢物的定量。 NMR 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，但可以有效地量化这些峰并充满信心 将它们分配给真正的代谢物仍然是一个重大挑战。 NMR 的标准方法 代谢组学就是检测1H，因为它既丰富又敏感。 1H NMR 的问题在于 峰经常重叠，使得可靠的识别和定量变得困难。我们开发了新的 使用 NMR 检测 13C 进行代谢组学的方法，包括天然丰度和同位素 富集，以利用由于 13C 和 13C 的大光谱色散而减少峰重叠的优势 化学位移与代谢物的更强大的数据库匹配。 13C NMR 的主要局限性 代谢组学是敏感性。我们建议开发由以下材料制成的 5 毫米 13C 优化 800 MHz NMR 探头： 高温超导体（HTS）将至少将代谢组学样品的灵敏度提高 比当前可用的容量高出 3 倍。灵敏度的提高将减少测量时间 至少 9 倍，否则我们将能够检测浓度低 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 上实施 MHz NMR 波谱仪可提高灵敏度和通量。基于 13C 信号到目标值 对于 ASTM 标准 9000:1 的噪声，我们预计能够完全量化和识别高达约 130 约 2 小时内即可在生物流体（如人血清）中产生代谢物。我们还在开发分馏方法 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。我们应该能够描述 300 多个 5 倍 SPE 浓度的代谢物，或 10 倍 SPE 浓度的 450 种代谢物。该项目将大大改善 代谢组学的再现性、可靠性和生物信息内容。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实现技术。 目标 1) 开发将安装在 Bruker Avance III HD 上的 18.8 T 5-mm 13C 优化 HTS 探头 佐治亚大学复杂碳水化合物研究中心 (CCRC) 的核磁共振波谱仪。 目标 2) 使用 2 个具有定量 13C 1D 和 同时进行 1H 2D NMR 实验。 LC-SPE 将允许浓缩并与 MS 耦合。

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.