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

Abstract

Glycosphingolipids (GSLs) are a class of ubiquitous lipids characterized by a wide structural repertoire and a variety of functional implications. Importantly, altered levels have been correlated with different diseases, suggesting their crucial role in health. Conventional methods for the characterization and quantification are based on high-performance TLC (HPTLC) separation and comparison with the migration distance of standard samples or on MS. We set up and herein report the application of an ImagePrep method for glycosphingolipids qualitative and quantitative profiling through direct HPTLC-MALDI with particular application to wild-type and NEU3 sialidase-overexpressing C2C12 myoblasts. Lipids were analyzed by HPTLC, coupled with MALDI-TOF, and the resulting GSLs profiles were compared to the [³H]sphingolipids HPTLC patterns obtained after metabolic radiolabeling. GSLs detection by HPTLC-MALDI was optimized by testing different methods for matrix delivery and by performing quantitative analyses using serial dilutions of GSLs standards. Through this approach an accurate analysis of each variant of neutral and acidic GSLs, including the detection of different fatty-acid chain variants for each GSL, was provided and these results demonstrated that HPTLC-MALDI is an easy and high-throughput analytical method for GSLs profiling, suggesting its use for an early detection of markers in different diseases, including cancer and heart ischemia.

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

“…Optimized conditions for obtaining GSL spectra from TLC plates have been determined by Torretta et al () using an ImagePrep device from BrukerDaltonics. DHB was the chosen matrix and applied under a variety of conditions.…”

Quantitative surface scanning by Direct Analysis in Real Time mass spectrometry

“…The spatial resolution was demonstrated to be below 1 mm with a FWHM of 0.8 mm for EE. Contrarily to matrix‐assisted laser desorption/ionization (MALDI), desorption electrospray ionization (DESI), plasma‐assisted multiwavelength laser desorption ionization (PAMLDI), and infrared laser ablation metastable‐induced chemical ionization (IR‐LAMICI) with better spatial resolutions, desorption and ionization take place in the sheer flow of the DART gas stream, or even plasma at higher DART temperatures. Thus matrix signals (MALDI), distortion of the zone shape due to involved solvents (DESI) or additional laser shot equipment (PAMLDI and IR‐LAMICI) do not complicate this DART setup.…”

Labeled gangliosides: their synthesis and use in biological studies

“…). To this end, 3 H‐ or 14 C‐labeled glucosamine and galactose , mannosamine , serine , or sphingosine were provided to cells, and their incorporation into glycolipids was determined. Unfortunately, the resulting specific radioactivity is very low, and furthermore, the radioactive ganglioside mixture must be fractionated to give single homogeneous gangliosides .…”