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.