Slow-MAS NMR Metabolomics

描述（由申请人提供）：高分辨率魔角旋转（hr-MAS）核磁共振，样品旋转速率为几kHz或更高，已成为完整生物组织代谢谱分析的有力工具。然而，为了使MAS NMR在生物医学、临床和转化研究中得到广泛应用，有几个关键问题需要解决。首先，hr-MAS技术是破坏性的，因为与快速样品纺丝相关的离心力很大。其次，由于与快速纺丝相关的各种技术挑战，hr-MAS实验中的样本量被限制在~15至60毫升。我们的研究目标是开发一种非破坏性，高分辨率和高灵敏度的MAS-NMR方法，补充hr-MAS用于代谢组学研究。为实现这一目标，我们制定了两个具体目标。目标1：通过在500 MHz核磁共振光谱仪上使用慢速样品旋转，包括慢速MAS探针和转子位置同步慢速MAS脉冲序列，开发一种非破坏性的MAS NMR代谢组学技术。slow-MAS技术将能够对生物组织样品进行高分辨率和高灵敏度的代谢分析，样品的体积变化范围从200纳升（nL）到1000微升（1.0 cm3）或更多，使用单个探针。nL的能力将使通过对单个小型实验动物的持续调查，并最终对患者进行长期的微创组织活检和血液样本的跟踪代谢变化成为可能。微升到立方厘米的能力将满足对不同大小的完整生物组织进行代谢谱分析的广泛需求，从而实现对完整组织进行大规模代谢谱分析。我们已经成功地在300 MHz核磁共振光谱仪上进行了概念验证的实验，使用概念验证的慢mas核磁共振探针来证明我们提出的研究是正确的。目的2:nL慢- mas方法的应用。我们将利用慢- mas探针的nL特性，在8 - 16周龄的8只肥胖C57BL/6小鼠和8只正常C57BL/6小鼠（对照组）中，使用200 - 500 nL体积的最小创活检骨骼肌和血液样本，持续跟踪代谢变化，以确定可能与肥胖相关的代代物生物标志物。在生命采样结束时，

DESCRIPTION (provided by applicant): High resolution magic angle spinning (hr-MAS) NMR where a sample spinning rate of a few kHz or more is used has become a powerful tool for metabolic profiling of intact biological tissues. However, there are a few critical issues that nee be addressed in order for MAS NMR to be used widely in biomedical, clinical, and translational researches. First, hr-MAS technique is destructive due to the large centrifugal force associated with fast sample spinning. Second, the sample volume in an hr-MAS experiment is restricted to ~15 to 60 ml also due to a variety of technical challenges associated with fast spinning. The goal of our research is to develop a non-destructive, high resolution and high sensitivity MAS-NMR method that complements hr-MAS for metabolomics investigations. To reach our goal, we have formulated two specific Aims. Aim 1: Development of a non-destructive MAS NMR metabolomics technique by using slow sample spinning, including a slow-MAS probe and rotor position synchronized slow-MAS pulse sequences on a 500 MHz NMR spectrometer. The slow-MAS technology will be capable of high resolution and high sensitivity metabolic profiling on biological tissue samples with volume variable from as small as 200 nanoliters (nL) to as large as 1000 microliters (1.0 cm3) or more using a single probe. The nL capability will make it possible to follow the metabolic changes through a continued investigation on a single small laboratory animal, and ultimately on a patient, over a long period of time using minimally invasive tissue biopsy and blood samples. The micro-liter to cm3 capability will serve the wide spread need of metabolic profiling on intact biological tissues of variable sizes, thus enabling large scale metabolic profiling on intact tissues. We have successfully performed concept- proven experiment on a 300 MHz NMR spectrometer using a concept-proven slow-MAS NMR probe to justify our proposed research. Aim 2: Application of the nL slow-MAS method. We will apply the nL feature of the slow-MAS probe to continuously follow the metabolic changes using minimal invasive biopsy skeletal muscle and blood samples of 200 to 500 nL in volume on 8 obese C57BL/6 mice, and 8 normal C57BL/6 mice (controls) over ages 8 to 16 weeks to identify possible metabolite biomarkers that are related to obesity. At the end of the in life sampling, the mice will be sacrificed and whole organs will be studied using the cm3 feature of the probe. We will also carry out slow-MAS metabolomics studies on artery excised from an obese- accelerated atherosclerosis mouse model. Obesity has become a recognized risk factor for a variety of metabolic disorders, including in particular atherosclerotic cardiovascular diseases. However, the disordered metabolic pathways that contribute to obesity-accelerated atherosclerosis are not well established. This is mainly due to the difficulties of direct metaboli profiling on very small amount of tissue samples. Our nL slow- MAS capability will make the metabolic profiling on the intact artery excised from a diet-induced obese + atherosclerosis mouse model possible.