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Assaying Ceramide Synthase Activity In Vitro and in Living Cells Using Liquid Chromatography-Mass Spectrometry

  • Xin Ying Lim
  • Russell Pickford
  • Anthony S. DonEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1376)

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.

Key words

Ceramide Ceramide synthase Assay Mass spectrometry LC-MS Liquid chromatography CERS 

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Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Xin Ying Lim
    • 1
  • Russell Pickford
    • 2
  • Anthony S. Don
    • 1
    Email author
  1. 1.Prince of Wales Clinical School, Faculty of MedicineUniversity of New South WalesSydneyAustralia
  2. 2.Bioanalytical Mass Spectrometry FacilityUniversity of New South WalesSydneyAustralia

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