ANALYSIS OF SPHINGOLIPIDS FOR LIPIDOMICS USING MALDI AND LC-ESI MASS SPECTROMETRY

PESCIO LG; STERIN SPEZIALE N; PAVÁN, CH; FRANCISCO, MN

Congreso; The LV Annual SAIB Meeting and XIV PABMB Congress; 2019

Sphingolipids are a complex family of bioactive lipids that are involved in many important processes such as cell signaling, cell differentiation, proliferation, cell survival and apoptosis. It is demonstrated that the different subspecies of sphingolipids containing different fatty acids have distinct functions. The most powerful and specific techniques for the identification and quantification of the molecular species of sphingolipids are chromatography combined with mass spectrometry. In this work we analyzed sphingolipid standards and sphingolipid extracts from epithelial cell cultures by TLC - MALDI and LC ? ESI MS, and compared both methodologies. The TLC was developed in two solvent systems for the separation of ceramide (Cer) and glucosylceramide (GlcCer) and stained with primuline. The bands were scraped off the TLC, extracted, mixed with the matrix (2,5-dihydroxylbenzoic acid, DHB) and analyzed using a 4800 MALDI TOF/TOF? analyzer. We detected different subspecies of Cer, GlcCer, LacCer and sphingomyelin: d18:1/C16:0, d18:1/C18:0, d18:1/C20:0, d18:1/C22:0, d18:1/C24:1 and d18:1/C24:0. For ESI analysis, the sphingolipid extracts were separated by LC using different mobile phases and reverse phase columns. The equipment used was a LCQ-Duo ESI -IT. The comparison of both techniques showed that MALDI TOF TOF MS analyses required short times of fine tuning and detection, but need prior separation of the sphingolipids present in the lipid extract by TLC, since each revealed band was scraped, extracted from the silica and analyzed by MALDI TOF TOF to avoid signal suppression. Moreover, the spectra showed severe matrix background and the presence of multiple adducts that complicates quantitation. On the other hand, LC - ESI MS simplified the procedure for lipid analysis, since the complete lipid extract was injected directly into the column, allowing the simultaneous separation and detection in the same assay. However, the fine tuning of LC ? ESI MS was more laboriously because of different variables must be adjust: mobile phase composition and additives, type of column, flow rate, type and optimization of detection and fragmentation conditions. Furthermore, the results obtained from LC?ESI MS were more complex than MALDI MS results, complicating mass spectral interpretation. The selection of the technique for sphingolipid analyses depends on the complexity of the sample and the objective of the investigation.