High Resolution Metabolomics

描述（申请人提供）：代谢组学正在成为研究生物系统的重要方法。同时测量许多代谢物导致了疾病生物标志物、药物作用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大部分代谢组的能力方面受到限制。因此，代谢组中的许多变化无法通过当前技术检测到。高压液相色谱-质谱联用（HPLC-MS）是代谢组学的重要方法。在这项工作中，我们寻求开发新的HPLC方法，这将大大提高复杂混合物的分辨率，并将这些方法应用于代谢组学。HPLC的分离能力随着柱长和固定相颗粒直径的减小而增加。改变HPLC柱的这些尺寸也增加了泵送移动的相通过柱所需的压力。HPLC传统上被限制在约8，000 psi（lbs/in 2），但先进的商业系统可以产生高达19，000 psi。这些“超”高压LC（UHPLC）系统的应用已经在检测复杂混合物中的代谢物的能力方面产生了显著的改进;然而，这些系统仍然远远达不到解析常见生物样品中的所有代谢物的目标。我们将开发具有100，000 psi能力和兼容LC柱的UHPLC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法来测量宽极性范围内的代谢物。我们假设，新技术将允许测量给定样品中比目前可能的多数千种代谢物。新方法还应提高复杂样品中代谢物检测的灵敏度和重现性。新的方法将用于确定参与脂肪形成模型的代谢途径，适应高运动能力和糖尿病并发症。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.