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

Abstract

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.

Application of direct HPTLC‐MALDI for the qualitative and quantitative profiling of neutral and acidic glycosphingolipids: The case of NEU3 overexpressing C2C12 murine myoblasts

“…The quantitative information available from MALDI mass spectra strongly depends on the homogenous distribution of the matrix, nevertheless, there are an increasing number of papers showing that accurate matrix deposition, the use of standards, and calibration curves can provide quantitative information . To demonstrate the applicability of HPTLC‐MALDI to quantitative analyses, a comparison with worldwide accepted methods based on radiolabeled precursor of sphingolipid species is mandatory, thus calibration curves of a neutral glycosphingolipid (Gb3Cer) and of a ganglioside (GM3) were constructed in triplicate.…”

“…These results demonstrate that HPTLC‐MALDI provides the exact monoisotopic mass overcoming the low resolution of HPTLC. In addition, by providing a direct analysis, it overcomes the transfer typical of TLC‐Blot‐MALDI or the silica scraping , reducing analytical steps, variability, and, possibly, improving the number of identified species. Peaks resolution over 3000 and 8000 for negative and positive mode, respectively, combined with Rf, allowed a precise analysis of GSLs composition.…”

“…Nevertheless, considering blank sample analyses and run times, its coupling with LC generated longer acquisition times than direct methods. Recently, to increase the sensitivity of GSLs detection, a PrimaDrop sample preparation prior to MALDI‐TOF acquisition has been introduced as a tool able to provide relative quantitative data on globotetra‐hexosylceramide (Gb4Cer) comparable with UPLC‐ESI‐MS/MS methodology . By this indirect method, plates were sprayed with primuline and bands were scraped off, dissolved in a solvent of choice and loaded on a ground steel target.…”

Analysis of carbohydrates and glycoconjugates by matrix‐assisted laser desorption/ionization mass spectrometry: An update for 2013–2014

“…In order to develop a quantitative assay for globotetraosylceramides (Gb4Cer) in a rat model of polycystic kidney disease, Ruh et al () first separated the compound by TLC and then used a new method termed “PrimaDrop” to analyze it by MALDI‐TOF with bradykinin as the internal standard. The PrimaDrop method involved evaporation of the sample on the MALDI target and drying rapidly with a hair dryer.…”

Mass‐spectrometry‐based lipidomics

“…Moreover, it is difficult to couple TLC with MS detection to improve its sensitivity. Usually, TLC can be coupled off‐line to MALDI‐MS . GC is a powerful chromatographic separation method that has high separation efficiency, making it suitable for the separation of volatile compounds.…”