Qualitative and quantitative measurements of sphingolipids by mass spectrometry

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.