Toward ‘Omic Scale Metabolite Profiling: A Dual Separation–Mass Spectrometry Approach for Coverage of Lipid and Central Carbon Metabolism
Year: 2013
Type: article
Abstract: Although the objective of any 'omic science is broad measurement of its constituents, such coverage has been challenging in metabolomics because the metabolome is comprised of a chemically diverse set of small molecules with variable physical properties. While extensive studies have been performed to identify metabolite isolation and separation methods, these strategies introduce bias toward lipophilic or water-soluble metabolites depending on whether reversed-phase (RP) or hydrophilic interaction liquid chromatography (HILIC) is used, respectively. Here we extend our consideration of metabolome isolation and separation procedures to integrate RPLC/MS and HILIC/MS profiling. An aminopropyl-based HILIC/MS method was optimized on the basis of mobile-phase additives and pH, followed by evaluation of reproducibility. When applied to the untargeted study of perturbed bacterial metabolomes, the HILIC method enabled the accurate assessment of key, dysregulated metabolites in central carbon pathways (e.g., amino acids, organic acids, phosphorylated sugars, energy currency metabolites), which could not be retained by RPLC. To demonstrate the value of the integrative approach, bacterial cells, human plasma, and cancer cells were analyzed by combined RPLC/HILIC separation coupled to ESI positive/negative MS detection. The combined approach resulted in the observation of metabolites associated with lipid and central carbon metabolism from a single biological extract, using 80% organic solvent (ACN:MeOH:H2O 2:2:1). It enabled the detection of more than 30,000 features from each sample type, with the highest number of uniquely detected features by RPLC in ESI positive mode and by HILIC in ESI negative mode. Therefore, we conclude that when time and sample are limited, the maximum amount of biological information related to lipid and central carbon metabolism can be acquired by combining RPLC ESI positive and HILIC ESI negative mode analysis.
Source: Analytical Chemistry
DOI: 10.1021/ac401140h
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
Funders: U.S. Department of Energy, California Institute of Regenerative Medicine, National Institutes of Health, National Institute on Aging, National Heart, Lung, and Blood Institute, National Institute of Mental Health, National Institute on Drug Abuse, National Eye Institute, National Cancer Institute, National Institute of General Medical Sciences, National Institute of Environmental Health Sciences
Awards: G1307824525, G1427424561, G1900904079, Normalizing Breast Cancer Metabolism to Prevent Progression and Recurrence (PQ21), G2498822990, MOLECULAR ASSEMBLIES, GENES AND GENOMICS INTEGRATED EFFICIENTY (MAGGIE), University of California Berkeley Chemistry-Biology Interface Training Program, Chronic HIV Infection and Aging in NeuroAIDS (CHAIN) Center, G42916606, G4377405353, Griffin: Venous Thromboembolism and mass spectrometry-based untargeted metabolomi, DEVELOPING THE UNTARGETED METABOLOMIC WORKFLOW FOR HIGH-THROUGHPUT ANALYSES, G5292805843, G5959837446, Adult Stem Cells for Therapy of Visual Disorders, Proteomic Strategies for AIDS and Drug Abuse - HIV and METH CNS Synergy, G71690937, G9000346257
OpenAlex ID: W2013233148