High Resolution Metabolomics

描述(由申请人提供)：代谢组学正在成为研究生物系统的一种重要方法。对多种代谢物的同时测量导致了疾病生物标志物、药物效应和细胞功能机制的重要发现。尽管代谢组学取得了成功，但目前的分析方法在测量代谢组的很大一部分方面的能力有限。因此，目前的技术无法检测到代谢组中的许多变化。高效液相色谱-质谱联用法是代谢组学研究的重要方法。在这项工作中，我们寻求开发新的高效液相色谱方法，以极大地提高对复杂混合物的拆分能力，并将这些方法应用于代谢组学。高效液相色谱的拆分能力随柱长和固定相颗粒直径的减小而增大。改变高效液相色谱柱的这些尺寸也增加了通过柱泵送流动相所需的压力。传统上，高效液相被限制在约8,000磅/平方英寸(lbs/in2)，但先进的商业系统可以产生高达19,000磅/平方英寸的能量。这些“超高压LC”系统的应用在检测复杂混合物中代谢物的能力方面取得了显著的进步，但这些系统仍然远远不能解决普通生物样品中所有代谢物的分离目标。我们将开发具有100,000磅/平方英寸容量和兼容LC色谱柱的超高效液相系统。我们将研究在这些极端压力下使用反相和亲水相互作用液相色谱来测量广泛的极性范围内的代谢物。我们假设，新技术将允许在一个给定的样本中测量比目前可能的更多的数千种代谢物。新方法还将提高复杂样品中代谢物检测的灵敏度和重现性。这些新方法将被用于识别脂肪生成、适应高运动能力和糖尿病并发症模型中涉及的代谢途径。此外，新开发的仪器和方法将被纳入国家代谢组学中心，以确保这项新技术的广泛影响。

DESCRIPTION (provided by applicant): Metabolomics is emerging as an important approach to study biological systems. Measurement of many metabolites at one time has led to important discoveries of disease biomarkers, drug effects, and mechanism of cellular function. Despite the success of metabolomics, current analytical methods are limited in their ability to measure a large fraction of the metabolome. As a result, many changes in the metabolome are not detected by current techniques. High pressure liquid chromatography coupled to mass spectrometry (HPLC-MS) is a prominent method for metabolomics. In this work, we seek to develop new approaches to HPLC that will vastly improve the resolving power for complex mixtures and apply these methods to metabolomics. The resolving power of HPLC increases with column length and with decreasing diameter of stationary phase particles. Changing these dimensions of the HPLC column also increases the pressure required to pump mobile phase through the column. HPLC has traditionally been limited to about 8,000 psi (lbs/in2), but advanced commercial systems can generate up to 19,000 psi. Application of these "ultra" high pressure LC (UHPLC) systems has yielded significant improvement in the ability to detect metabolites in complex mixtures; however, these systems still fall far short of the goal of resolving all metabolites in common biological samples. We will develop UHPLC systems with capability of 100,000 psi and compatible LC columns. We will investigate use of both reversed phase and hydrophilic interaction liquid chromatography at these extreme pressures to measure metabolites across a broad polarity range. We hypothesize that the new technology will allow measurement of thousands of more metabolites in a given sample than currently possible. The new method should also enhance sensitivity and reproducibility of metabolite detection in complex samples. The new methods will be used to identify metabolic pathways involved in models of adipogenesis, adaptation to high exercise capacity, and diabetic complications. Further, newly developed instrumentation and methods will be incorporated into a national center for metabolomics to ensure widespread impact of the new technology.