Observation of different ceramide species from crude cellular extracts by normal-phase high-performance liquid chromatography coupled to atmospheric pressure chemical ionization mass spectrometry.
Normal-phase high-performance liquid chromatography (NP-HPLC) coupled to atmospheric pressure chemical ionization mass spectrometry (APCI-MS) allows qualitative analysis of endogenous ceramide and dihydroceramide species from crude lipid extracts utilizing chromatographic methods readily adaptable from commonly used thin layer chromatography (TLC) conditions. Qualitative information for the species comes from observation of differences in chromatographic and mass spectrometric behavior between species. Application to the analysis of ceramide and dihydroceramide from various cell lines is demonstrated. The results show the species profile in each cell line to be unique despite growth under identical conditions. The results from APCI-MS analysis corroborate and enhance information acquired from use of the diacylglycerol kinase assay for total ceramide measurement. This technique readily allows the previously difficult distinction between ceramide and dihydroceramide species.
Pettus
Benjamin J
BJ
Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, SC 29425, USA.
Bielawska
Alicia
A
Kroesen
Bart-Jan
BJ
Moeller
Peter D R
PD
Szulc
Zdzislaw M
ZM
Hannun
Yusuf A
YA
Busman
Mark
M
D001274:Atmospheric Pressure; D002457:Cell Extracts / Q000737:chemistry*; D002518:Ceramides / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D019852:Diacylglycerol Kinase / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D014407:Tumor Cells, Cultured
D002457:Cell Extracts; D002518:Ceramides; C109343:dihydroceramide; D019852:Diacylglycerol Kinase