Quantitative characterization of tissue globotetraosylceramides in a rat model of polycystic kidney disease by PrimaDrop sample preparation and indirect high-performance thin layer chromatography-matrix-assisted laser desorption/ionization-time-of-flight-mass spectrometry with automated data acquisition.

Glycosphingolipids (GSL) have been associated with a variety of diseases, including cancer and autosomal dominant polycystic kidney disease (ADPKD). In contrast to glucosylceramide and gangliosides, alterations in complex neutral GSLs such as globotetraosylceramide (Gb4Cer) have not been investigated in ADPKD yet, and mass spectrometry analysis of Gb4Cer from tissue extracts remains challenging. To this end, we introduce PrimaDrop as an improved and widely applicable sample preparation method for automated matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) analysis of lipid extracts, which promotes homogeneous cocrystallization and enables relative quantification by indirect thin layer chromatography (TLC)-MALDI-time-of-flight (TOF)-MS against an internal bradykinin standard. Application of the method for detailed investigation of Gb4Cer isoforms in kidneys of an ADPKD rat model revealed increased levels of sphingoid base-containing isoforms in cystic kidneys, whereas changes were subtle for Gb4Cer-containing phytosphingoid bases. We furthermore established an absolute LC-ESI-MS/MS quantification method and demonstrate that absolute quantities of Gb4Cer correlate well with relative quantities obtained by indirect TLC-MALDI-TOF-MS. Taken together, our study proposes an effective sample preparation method for automated analysis of lipid extracts and TLC eluates and suggests that indirect high-performace (HP)TLC-MALDI-TOF-MS with automated data acquisition is a viable option for analysis of neutral glycosphingolipids and that Gb4Cer may play a role in the pathogenesis of ADPKD.