Multiplexed UPLC-MS/MS System for Advanced Target Metabolomics

 描述(申请人提供)：位于西雅图华盛顿大学的西北代谢组学研究中心(NW-MRC)是太平洋西北地区最大的综合代谢图谱实验室。我们已经开发了一个强大的、可重复使用的靶向质谱学平台，可以测量200多种水溶液代谢物，需求量非常大。该平台基于与安捷伦1260双液相色谱泵相连的AB-SCHEX API5500 Qtrap质谱仪，该双液相色谱泵使用HILIC柱在两个相同的条件下进行正负电离，分离水中的代谢物。由于其良好的测量重复性和稳健性，该方法引起了基础和临床研究的高度兴趣。例如，自2014年初以来，我们已经分析了来自该系统60多个合作者的3500多个生物样本，并在10篇同行评议文章中报告了结果。在这一点上，对系统的需求超出了我们的能力，随着我们经历的强劲增长，我们预计运行样本的延迟会越来越长。此外，我们希望在我们的检测中添加靶向脂质组学，但没有时间用于方法开发或在该平台上运行额外的脂类。最后，我们开发了一种新的代谢组学方法，全局优化靶向(GOT)-MS，它结合了全局分析和靶向分析的优点，并承诺非常广泛的代谢组覆盖范围和高重复性。然而，我们的AB-SCHEX系统是我们唯一一个可以对200多个分子进行定向测量的系统，具有良好的长期稳定性。鉴于对该系统的高需求，我们迫切需要增加一个更通用、更灵敏的LC-MS/MS系统，以用于高级靶向代谢组学。为了进一步增强靶向代谢组学能力，我们建议安装最先进的UPLC-MS/MS系统，能够在四个分析柱上同时分离，然后进行高灵敏度的靶向MS采集。每个色谱柱将独立连接到一个4端口进样自动取样器，该进样自动取样器将允许在四种不同的层析条件下进行分离。这一平台的主要好处将是：(I)测量代谢物的覆盖范围更广，因为我们将在我们的目标分析中添加数百种脂质；(Ii)吞吐量能力的显著增强，这反过来将消除分析周转时间的延误；(Iii)可用于方法开发的额外时间。因此，我们将能够开发和利用我们新的GOT-MS方法来改进代谢物测量。建议的系统将用于合作研究项目至少75%的运行时间，它的广泛使用将导致更好地理解与疾病发展、生物标记物发现和系统生物学研究相关的代谢过程。

 DESCRIPTION (provided by applicant): The Northwest Metabolomics Research Center (NW-MRC) at the University of Washington in Seattle is the largest comprehensive metabolic profiling laboratory in the Pacific Northwest Region. We have developed a robust and reproducible targeted mass spectrometry platform that measures more than 200 aqueous metabolites and is in very high demand. This platform is based on an AB-Sciex API5500 Qtrap mass spectrometer coupled to Agilent 1260 dual liquid chromatography pumps that separate aqueous metabolites using HILIC columns under two identical conditions for both positive and negative ionization. Due to its excellent measurement reproducibility and robustness, this assay has attracted a very high interest from basic and clinical researches. For example, since the beginning of 2014, we have analyzed over 3,500 biological samples from more than 60 collaborators on this system, and the results have been reported in 10 peer-reviewed articles. At this point, demand for the system is exceeding our capacity, and with the strong growth we have experienced, we anticipate longer and longer delays in running samples. In addition, we would like to add targeted lipidomics to our assay, but do not have instrument time to devote for method development or for running the additional lipids on this platform. Finally, we have developed a new approach for metabolomics, globally optimized targeted (GOT)-MS, which combines the benefits of global and targeted analysis and promises very broad metabolome coverage with high reproducibility. However, our AB-Sciex system is the only one we have that can perform targeted measurements of over 200 molecules with good long term stability. Given the high demand for this system, we greatly need the addition of a more versatile and sensitive LC-MS/MS system for advanced targeted metabolomics. To further enhance targeted metabolomics capabilities we propose to install a state-of-the-art multiplexed UPLC- MS/MS system capable of performing simultaneous separations on four analytical columns followed by high sensitive targeted MS acquisition. Each column will be independently connected to a 4-port injection auto- sampler, which in turn will allow separations under four different chromatography conditions. The major benefits of this platform will be: (i) much broader coverage of measured metabolites since we will add hundreds of lipids to our targeted assay; (ii) significant enhancement in the throughput capabilities, which in turn will eliminate delays in analysis turnaround time; (iii) additional time available for method development. As a result, we will be able to develop and utilize our new GOT-MS methodology to improve metabolite measurements. The proposed system will be available for collaborative research projects at least 75% of the run time, and its extensive utilization will lead to a better understanding of metabolic processes associated with disease development, biomarker discovery, and systems biology research.