ALBERT

Description:    Sphingolipids are an important class of compounds that regulate signal transduction and other vital cellular processes. Herein, we report sensitive normal and reversed phase LC–MS/MS methods for quantitation of multiple sphingolipid classes. In the normal-phase ESI/MS/MS method, a high content of organic solvents was utilized, which, although it included hexane, ethyl acetate, acetonitrile containing 2% methanol, 1–2% acetic acid, and 5 mM ammonium acetate, resulted in a very efficient electrospray ionization of the ceramides (Cers) and hexosylceramides (MHCers). Three normal-phase LC–MS/MS methods using segmented phases were developed to specifically target Cers, MHCers, or sphingomyelins (SMs). This segmentation scheme increases the number of data points acquired for a given analyte and enhances the sensitivity and specificity of the measurements. Nine separate reversed phase chromatography methods were developed for the three classes of compounds. These assays were used for comparing the levels of Cers, SMs, and MHCers from mouse embryonic fibroblast (pMEF) and human embryonic kidney (HEK293) cells. These findings were then compared with the reported data from RAW264.7 mouse macrophage cells, BHK21 hamster cells, and human plasma and serum samples. The analysis of cell lines, using both normal and reversed phase chromatography, revealed discrimination based on the type of chromatography chosen, while sphingolipid assays of samples containing different amounts of protein showed different results, even after normalizing for protein content. Also, LC/MS/MS profiles were provided for the classes and individual compounds so that they could be used as “molecular profiles” for class or individual sample analysis. Content Type Journal Article Category Original Article Pages 209-226 DOI 10.1007/s11745-011-3633-2 Authors M. Athar Masood, Laboratory of Proteomics and Analytical Technologies, SAIC-Frederick, Inc., National Cancer Institute at Frederick, Frederick, MD 21702-1201, USA Raghavendra P. Rao, Laboratory of Cell and Developmental Signaling, National Cancer Institute at Frederick, Frederick, MD 21702-1201, USA Jairaj K. Acharya, Laboratory of Cell and Developmental Signaling, National Cancer Institute at Frederick, Frederick, MD 21702-1201, USA Josip Blonder, Laboratory of Proteomics and Analytical Technologies, SAIC-Frederick, Inc., National Cancer Institute at Frederick, Frederick, MD 21702-1201, USA Timothy D. Veenstra, Laboratory of Proteomics and Analytical Technologies, SAIC-Frederick, Inc., National Cancer Institute at Frederick, Frederick, MD 21702-1201, USA Journal Lipids Online ISSN 1558-9307 Print ISSN 0024-4201 Journal Volume Volume 47 Journal Issue Volume 47, Number 2