Quantitation of Multiple Sphingolipid Classes Using Normal and Reversed‐Phase LC–ESI–MS/MS: Comparative Profiling of Two Cell Lines

• DOI: 10.1007/s11745-011-3633-2
• Publication Date: 2011-11-28T09:33:08Z
• Authors: M. Athar Masood; Raghavendra P. Rao; Jairaj K. Acharya; Josip Blonder; Timothy D. Veenstra
• Keywords: Sphingolipid

Sphingolipids are an important class of compounds that regulate signal transduction and other vital cellular processes. Herein, we report sensitive normal reversed phase LC-MS/MS methods for quantitation multiple sphingolipid classes. In the normal-phase ESI/MS/MS method, a high content organic solvents was utilized, which, although it included hexane, ethyl acetate, acetonitrile containing 2% methanol, 1-2% acetic acid, 5 mM ammonium resulted in very efficient electrospray ionization ceramides (Cers) hexosylceramides (MHCers). Three using segmented phases were developed to specifically target Cers, MHCers, or sphingomyelins (SMs). This segmentation scheme increases number data points acquired given analyte enhances sensitivity specificity measurements. Nine separate chromatography three classes compounds. These assays used comparing levels SMs, MHCers from mouse embryonic fibroblast (pMEF) human kidney (HEK293) cells. findings then compared with reported RAW264.7 macrophage cells, BHK21 hamster plasma serum samples. The analysis cell lines, both chromatography, revealed discrimination based on type chosen, while samples different amounts protein showed results, even after normalizing content. Also, LC/MS/MS profiles provided individual so they could be as "molecular profiles" sample analysis.