Addressing the analytical challenges of metabolomic studies: Increasing the throughput and reducing the complexity.

Adam M King

Metabolomics is a field of research focused on investigating metabolic mechanisms in response to health and disease, whether by analysing known compounds or novel metabolites as markers. Due to the diversity of analytes measured during these studies, the flexible analytical tool of liquid chromatography-mass spectrometry (LC-MS) is one of the most widely utilized techniques, with methodologies employing high resolution instruments, providing broad compound coverage and accurate mass for identification of unknowns. Many metabolomic projects require large sample numbers, sometimes into the thousands, providing statistical power to the results. This requires a balance between the time taken to analyse each sample and produce sufficient detail. Thus, many studies take weeks to complete, leading to instrument drift and batch effects. In this study, the challenge of increasing the throughput of large scale metabolomic studies was addressed by investigating rapid chromatographic separation techniques. Conventional chromatographic methods used in metabolomics require long acquisitions, between 10 and 20 min per sample, increasing pressure on instrument resources and data variation. Conventional methods were geometrically scaled, using analytical columns with reduced internal diameters to shorten sample acquisition times by 60 %. Furthermore, an orthogonal separation technique, ion mobility, was investigated whereby compound feature counts increased by 50 % and co-eluting features were resolved. The rapid LC-MS methods developed for the analysis of small polar 7 molecules and dedicated lipid analysis provide a suite of methods which were applied for the analysis of human serum samples derived from a prostate cancer study. A total of 350 human serum samples were analysed using three rapid ion mobility mass spectrometry methodologies. The stability and coverage of each method was assessed, and the complete sample analysis was completed in 1/3 of the time when compared with conventional approaches. With the demonstrated benefit of high-resolution mass spectrometry and ion mobility to increase feature counts and resolve co-eluting species , further work was conducted to examine advanced cyclic ion mobility for the separation of structural isomers and location of metabolite conjugation, highlighting the benefit of this novel technology. The cyclic multi-pass separation function enabled the resolution of multiple forms of sulphated tyrosine and the identification of sulfation sites. Furthermore, plasma analysis was carried out using a novel multi-reflecting time-of-flight instrument where the improved mass resolution of 224,770 FWHM at m/z = 758.5705 assisted in distinct separation of two co-eluting lipid features (PC (34:2) and PC (34:1)) and the detection of a fine isotopic distribution resulting from isotopes of carbon and potassium. This study has highlighted the benefits of high-throughput chromatography for increasing sample throughput and reducing instrument variability which, combined with ion mobility separation and advances in high-end mass analysers, can enhance discovery metabolomic studies and improve confidence in metabolite identification.

Addressing the analytical challenges of metabolomic studies: Increasing the throughput and reducing the complexity.

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