title: Improvements to define mitochondrial metabolomics using nonaqueous fractionation.
abstract: Defining metabolite abundance and resulting fractional isotope enrichments, within and between cellular compartments, still remain a major challenge in modern plant biochemistry. Optimized protocols for rapid isolation of mitochondria (e.g., silicone oil centrifugation or membrane filters) or visualization of metabolites/metabolic states (e.g., fluorescence resonance energy transfer (FRET) or redox-sensitive fluorescent markers (roGFP)) have significantly improved and expanded our knowledge regarding mitochondrial metabolism. However, the application of nonaqueous fractionation (NAQF) to separate and quantify metabolites across subcellular compartments remains popular as a nontargeted, validated approach towards studying metabolism, and provides a top-down overview of metabolite distribution across the majority of the subcellular compartments in a single preparation. Unfortunately, of all the organelles resolved using this method, the mitochondrion still remains the most poorly defined. Here, the development and suggested improvements to resolve the mitochondrial metabolome are described.
authors.affiliation: Institute for Plant Biotechnology, Stellenbosch University, Private Bag X1, Matieland, 7602, South Africa.
authors.forename: Richard
authors.initials: R
authors.lastname: Fly
authors.forename: James
authors.initials: J
authors.lastname: Lloyd
authors.forename: Stephan
authors.initials: S
authors.lastname: Krueger
authors.forename: Alisdair
authors.initials: A
authors.lastname: Fernie
authors.forename: Margaretha J
authors.initials: MJ
authors.lastname: van der Merwe
mesh_terms: D002458:Cell Fractionation / Q000379:methods*; D002498:Centrifugation / Q000379:methods; D013058:Mass Spectrometry / Q000379:methods; D055442:Metabolome; D055432:Metabolomics / Q000379:methods*; D008928:Mitochondria / Q000201:enzymology / Q000378:metabolism*; D010944:Plants / Q000201:enzymology / Q000378:metabolism*