Advanced Multiplexed UPLC-MS/MS System for Target Metabolomics

About This Grant

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 370 aqueous metabolites and is in very high demand. This platform is based on an AB-Sciex API 6500+ mass spectrometer coupled to Shimadzu NexeraXR LC-20 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 researchers. For example, over the past 3 years, we have analyzed over 14,000 biological samples from more than 60 collaborators on this system. Since we installed this system in 2017, which was funded by a successful S10 grant application, the results have been reported in 37 peer-reviewed articles. At this point, demand for the system exceeds 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 reversed phase chromatography to our assay in order to increase metabolite coverage, but we do not have the capacity to increase our overall analysis time given the large number of samples we currently run. Our AB-Sciex 6500+ system is the only one we have that can perform targeted measurements of over 370 aqueous molecules with high sensitivity and good long-term stability. While we have a Sciex 5500 system, it is dedicated to performing targeted lipidomics and oxylipin analysis. Given the high demand for our 6500 system and its targeted aqueous assay, 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 highly 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 the higher sensitivity and 4 chromatography columns will allow us to add hundreds of additional metabolites 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. 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.