title: Assaying Ceramide Synthase Activity In Vitro and in Living Cells Using Liquid Chromatography-Mass Spectrometry.
abstract: Sphingolipids are one the major lipid families in eukaryotes, incorporating a diverse array of structural and signaling lipids such as sphingomyelin and gangliosides. The core lipid component for all complex sphingolipids is ceramide, a diacyl lipid consisting of a variable length fatty acid linked through an amide bond to a long chain base such as sphingosine or dihydrosphingosine. This reaction is catalyzed by a family of six ceramide synthases (CERS1-6), each of which preferentially catalyzes the synthesis of ceramides with different fatty acid chain lengths. Ceramides are themselves potent cellular and physiological signaling molecules heavily implicated in diabetes and neurodegenerative diseases, making it important for researchers to have access to sensitive and accurate assays for ceramide synthase activity. This chapter describes methods for assaying ceramide synthase activity in cell or tissue lysates, or in cultured cells (in situ), using liquid chromatography-tandem mass spectrometry (LC-MS/MS) as the readout. LC-MS/MS is a very sensitive and accurate means for assaying ceramide synthase reaction products.
authors.affiliation: Prince of Wales Clinical School, Faculty of Medicine, University of New South Wales, Sydney, NSW, 2052, Australia.
authors.forename: Xin Ying
authors.initials: XY
authors.lastname: Lim
authors.affiliation: Bioanalytical Mass Spectrometry Facility, University of New South Wales, Sydney, NSW, 2052, Australia.
authors.forename: Russell
authors.initials: R
authors.lastname: Pickford
authors.affiliation: Prince of Wales Clinical School, Faculty of Medicine, University of New South Wales, Sydney, NSW, 2052, Australia. anthonyd@unsw.edu.au.
authors.forename: Anthony S
authors.initials: AS
authors.lastname: Don
mesh_terms: D000818:Animals; D002518:Ceramides / Q000378:metabolism; D002853:Chromatography, Liquid / Q000379:methods; D004789:Enzyme Activation; D066298:In Vitro Techniques; D051379:Mice; D010088:Oxidoreductases / Q000378:metabolism*; D053719:Tandem Mass Spectrometry / Q000379:methods
chemical_list: D002518:Ceramides; D010088:Oxidoreductases; C108138:dihydroceramide desaturase
keywords: Assay; CERS; Ceramide; Ceramide synthase; LC-MS; Liquid chromatography; Mass spectrometry