Sphingolipidomics: High-throughput, structure-specific, and quantitative analysis of sphingolipids by liquid chromatography tandem mass spectrometry

• Published in: Methods (San Diego, Calif.) Calif.), 2005-06, Vol.36 (2), p.207-224
• Main Authors: Merrill, Alfred H., Sullards, M. Cameron, Allegood, Jeremy C., Kelly, Samuel, Wang, Elaine
• Format: Article
• Language: English
• Subjects: Animals; Bioactive sphingolipids; Chromatography, Liquid - methods; Glycosphingolipids - chemistry; Humans; Lipid mass spectrometry; Lipidomics; Lipids - chemistry; Mass Spectrometry - methods; Models, Chemical; Spectrometry, Mass, Electrospray Ionization; Sphingolipid analysis; Sphingolipid metabolism; Sphingolipids - chemistry; Sphingomyelins - chemistry; Sphingosine - analogs & derivatives; Sphingosine - chemistry; Time Factors
• ISSN: 1046-2023; 1095-9130
• DOI: 10.1016/j.ymeth.2005.01.009

Sphingolipids are a highly diverse category of compounds that serve not only as components of biologic structures but also as regulators of numerous cell functions. Because so many of the sphingolipids in a biological system are bioactive and are often closely related structurally and metabolically (for example, complex sphingolipids ↔ ceramide ↔ sphingosine ↔ sphingosine 1-phosphate), to understand the role(s) of sphingolipids in a given context one must conduct a “sphingolipidomic” analysis—i.e., a structure-specific and quantitative measurement of all of these compounds, or at least all members of a critical subset. Liquid chromatography tandem mass spectrometry (LC MS/MS) is currently the only technology with the requisite structural specificity, sensitivity, quantitative precision, and relatively high-throughput capabilities for such analyses in small samples (∼10 6 cells). This review describes a series of protocols that have been developed for the relatively rapid analysis of all of the molecular species from 3-ketosphinganines through sphingomyelins and some glycosphingolipids (including all the compounds that are presently regarded as sphingolipid “second messengers”) using normal- and reverse-phase LC to separate isometric and isobaric species (such as glucosylceramides and galactosylceramides) in combination with triple quadrupole (for MS/MS) and hybrid quadrupole–ion trap (for MS 3) mass spectrometry. Also discussed are some of the issues remaining to be resolved in the analysis of the full sphingolipidome.