title: A complete workflow for high-resolution spectral-stitching nanoelectrospray direct-infusion mass-spectrometry-based metabolomics and lipidomics.
abstract: Metabolomic and lipidomic studies measure and discover metabolic and lipid profiles in biological samples, enabling a better understanding of the metabolism of specific biological phenotypes. Accurate biological interpretations require high analytical reproducibility and sensitivity, and standardized and transparent data processing. Here we describe a complete workflow for nanoelectrospray ionization (nESI) direct-infusion mass spectrometry (DIMS) metabolomics and lipidomics. After metabolite and lipid extraction from tissues and biofluids, samples are directly infused into a high-resolution mass spectrometer (e.g., Orbitrap) using a chip-based nESI sample delivery system. nESI functions to minimize ionization suppression or enhancement effects as compared with standard electrospray ionization (ESI). Our analytical technique-named spectral stitching-measures data as several overlapping mass-to-charge (m/z) windows that are subsequently 'stitched' together, creating a complete mass spectrum. This considerably increases the dynamic range and detection sensitivity-about a fivefold increase in peak detection-as compared with the collection of DIMS data as a single wide mass-to-charge (m/z ratio) window. Data processing, statistical analysis and metabolite annotation are executed as a workflow within the user-friendly, transparent and freely available Galaxy platform (galaxyproject.org). Generated data have high mass accuracy that enables molecular formulae peak annotations. The workflow is compatible with any sample-extraction method; in this protocol, the examples are extracted using a biphasic method, with methanol, chloroform and water as the solvents. The complete workflow is reproducible, rapid and automated, which enables cost-effective analysis of >10,000 samples per year, making it ideal for high-throughput metabolomics and lipidomics screening-e.g., for clinical phenotyping, drug screening and toxicity testing.
authors.affiliation: School of Biosciences, University of Birmingham, Birmingham, UK.
authors.forename: Andrew D
authors.initials: AD
authors.lastname: Southam
authors.affiliation: School of Biosciences, University of Birmingham, Birmingham, UK.
authors.forename: Ralf J M
authors.initials: RJ
authors.lastname: Weber
authors.affiliation: NERC Biomolecular Analysis Facility - Metabolomics Node (NBAF-B), School of Biosciences, University of Birmingham, Birmingham, UK.
authors.forename: Jasper
authors.initials: J
authors.lastname: Engel
authors.affiliation: School of Biosciences, University of Birmingham, Birmingham, UK.
authors.forename: Martin R
authors.initials: MR
authors.lastname: Jones
authors.affiliation: School of Biosciences, University of Birmingham, Birmingham, UK.|NERC Biomolecular Analysis Facility - Metabolomics Node (NBAF-B), School of Biosciences, University of Birmingham, Birmingham, UK.
authors.forename: Mark R
authors.initials: MR
authors.lastname: Viant
mesh_terms: D045744:Cell Line, Tumor; D006801:Humans; D050356:Lipid Metabolism; D055432:Metabolomics / Q000379:methods*; D036103:Nanotechnology / Q000379:methods*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*