Abstract
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 which are relatively insensitive and/or time consuming. Tandem mass spectrometry with its high selectivity and sensitivity is a very useful technique for detection of low abundant metabolites without prior purification or derivatization. In contrast to existing mass spectrometry methods, the developed 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 autosampler, resulting in a high throughput capability. The collision-induced fragmentation of CER produced, in addition to others, a characteristic fragment of m/z 264, making a precursor ion scan of 264 well suited for CER quantitation. Quantitation is achieved by use of a constant concentration of a non-naturally occurring internal standard C8-CER, together with a calibration curve established by spiking different concentrations of naturally occurring CER. The calibration curves showed linearity over a wide concentration range and sample volumes equivalent to 10 μg of cell protein corresponding to about 20,000 fibroblasts were sufficient for CER analysis. Moreover this assay showed a detection limit at the subpicomole level.▪In summary, this methodology enables accurate and rapid analysis of CER from small samples without prior separation steps, thus providing a useful tool for signal transduction research.—Liebisch, G., W. Drobnik, M. Reil, B. Trümbach, R. Arnecke, B. Olgemöller, A. Roscher, and G. Schmitz. Quantitative measurement of different ceramide species from crude cellular extracts by electrospray ionization tandem mass spectrometry (ESI-MS/MS). J. Lipid Res. 1999. 40: 1539–1546.
Highlights
Ceramide (CER) is an important signaling molecule involved in a variety of cellular processes, including differentiation, cell growth, and apoptosis
This dissociation pattern was confirmed for distinct CER species, varying in N-linked fatty acid moiety (2 –24 carbons length), and a solvent and ionization additive was selected to generate a maximum of positive ions
All precursor ion peaks obtained by the analysis of an unprocessed fibroblast lipid extract could be attributed to the mass of certain CER species
Summary
Ceramide (CER) is an important signaling molecule involved in a variety of cellular processes, including differentiation, cell growth, and apoptosis. Been developed for the detection of CER, which are independent of prior fluorescence derivatization of the analyte and avoid a potential source of error These techniques still require an initial separation step of the crude lipid extract by HPLC. A qualitative determination of distinct CER species from relatively crude lipid extract was described by Gu et al [17], who performed a precursor ion scan (PIS) for a specific CER fragment resulting from collision-induced dissociation (CID) This procedure still needed an initial purification of the lipid extract by silica gel column and, the sole addition of an unnatural CER species as internal standard did not allow quantitation
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