Ceramide profiling of complex lipid mixtures by electrospray ionization mass spectrometry

Ceramides and sphingoid bases are important intracellular second messengers that play a role in the regulation of cell growth, differentiation, and programmed cell death. Until now, quantitative and qualitative analysis of ceramide second messengers has been limited by a lack of analytical methods capable of detecting endogenous levels of, and differentiating between, individual ceramide species. Here we report the use of electrospray ionization tandem mass spectrometry for the qualitative and quantitative analysis of ceramides. Collision-induced fragmentation resulted in characteristic product ions for the sphingosine and dihydrosphingosine (sphinganine) head groups at m/z 264 and 282 and m/z 266 and 284, respectively, regardless of the length of the fatty acyl chains, with spectra being reproducible at concentrations as low as 25 nM (25 fmol/microliter). These reporter ions were used to detect both sphingosine- and sphinganine-based ceramides in complex mixtures using precursor ion scan analysis. We demonstrated the application of this method for profiling the composition of ceramides in a commercial preparation of bovine brain ceramides and, following minimal chromatographic separation, a lipid extract of cultured T cells. Furthermore, we easily detected relative differences in individual ceramide species levels by comparing the profiles of three related lymphocyte cell lines, Jurkat, U937, and WEHI 231. Finally, by the addition of a nonnaturally occurring internal standard, we show that the technique can be used to measure quantitative changes in ceramide levels in such biologically derived lipid preparations.