Chemical Probes of Sphingolipid Metabolizing Enzymes

  • Ingrid Nieves
  • Pol Sanllehí
  • José Luis Abad
  • Gemma Fabriàs
  • Josefina Casas
  • Antonio Delgado


Sphingolipids (SLs) serve the dual roles of acting as structural entities in cellular membranes as well as bioactive signaling molecules that modulate signal transduction. As the already immense database of identified bioactive SL subspecies continues to expand, the need for structure-specific identification and quantification continues to rise. The characterization and analysis of the sphingolipidome by mass spectrometry has advanced steadily over the last 20 years with the aid of improvements in technological advancements in instrumentation, coupled with optimization of lipid extraction methodologies, and an increasing library of available reference standards. Pivotal advances in sphingolipidomics include the adoption of soft ionization techniques, including electrospray ionization (ESI), tandem mass spectrometry (MS/MS), and matrix-assisted laser desorption ionization (MALDI), as well as the use of multiple reaction monitoring (MRM), all of which have aided in improving the quality of analysis of often complex lipid extracts from mammalian, yeast, and even plant cells. In this chapter we explore qualitative and quantitative mass spectrometry methods used for structural elucidation and quantitation of sphingolipids found in cells as well as tissues. Sections included here detail extraction and HPLC methodologies, in vitro labeling techniques, use of internal and calibration lipid standards for quantitation, and data analysis of sphingolipids derived from mammalian and yeast sources.


Enzyme activity Chemical probe Chemical reporter Fluorescence Fluorophore Sphingolipid Metabolism 









Ceramide 1-phosphate


Ceramide kinase


Ceramide synthase


Dihydroceramide desaturase












Glucosylceramide synthase








High throughput screening


Isobaric tags for relative and absolute quantification








Nile Red








Sphingosine kinases








Sphingomyelin synthases




Sphingosine 1-phosphate


Sphingosine 1-phosphate lyase


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Copyright information

© Springer International Publishing Switzerland 2015

Authors and Affiliations

  • Ingrid Nieves
    • 1
  • Pol Sanllehí
    • 1
    • 2
  • José Luis Abad
    • 1
  • Gemma Fabriàs
    • 1
  • Josefina Casas
    • 1
  • Antonio Delgado
    • 1
    • 2
  1. 1.Consejo Superior de Investigaciones Científicas (CSIC), Institut de Química Avançada de Catalunya (IQAC-CSIC), Departament de Química Biomèdica, Research Unit on Bioactive Molecules (RUBAM)BarcelonaSpain
  2. 2.Universidad de Barcelona (UB), Facultad de Farmacia, Unidad de Química Farmacéutica (Unidad Asociada al CSIC)BarcelonaSpain

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