Quantitative measurement of different ceramide species from crude cellularextracts by electrospray ionization tandem mass spectrometry (ESI-MS/MS)

Ceramide (CER) is an important signaling molecule involved in a variety of cellular processes, including differentiation, cell growth, and apoptosis, Currently, different techniques are applied for CER quantitation, some of w hich are relatively insensitive and/or time consuming, Tandem mass spectrom etry with its high selectivity and sensitivity is a very useful technique f or detection of low abundant metabolites without prior purification or deri vatization, In contrast to existing mass spectrometry methods, the develope d electrospray tandem mass spectrometry (ESI-MS/MS) technique is capable of quantifying different CER species from crude cellular lipid extracts. The ESI-MS/MS is performed with a continuous flow injection and the use of an a utosampler, resulting in a high throughput capability, The collision-induce d fragmentation of CER produced, in addition to others, a characteristic fr agment of m/z 264, making a precursor ion scan of 264 well suited for CER q uantitation. Quantitation is achieved by use of a constant concentration of a non-naturally occurring internal standard CS-CER, together with a calibr ation curve established by spiking different concentrations of naturally oc curring CER, The calibration curves showed linearity over a wide concentrat ion range and sample volumes equivalent to 10 mu g of cell protein correspo nding to about 20,000 fibroblasts were sufficient for CER analysis, Moreove r this assay showed a detection limit at the subpicomole level. In summary, this methodology enables accurate and rapid analysis of CER from small sam ples without prior separation steps, thus providing a useful tool for signa l transduction research.