HPTLC‐MALDI MS for (glyco)sphingolipid multiplexing in tissues and blood: A promising strategy for biomarker discovery and clinical applications

Abstract

Sphingolipids have hydrophilic and hydrophobic properties, different saturation and combination of the oligosaccharide chains and mass homology of species located in a narrow m/z region hampering their recognition. To target sphingolipids for diagnostic purposes, standardized methods for lipid extraction, quali- and quantitative assessments are required. In this study, HPTLC-MALDI MS was adopted to establish sphingolipid and glycosphingolipid profiles in muscle, brain and serum to create a database of molecules to be searched in the preclinical and clinical investigations. Specific protocols for lipid extraction were set up based on the characteristics of the tissue or/and fluids; this approach maximizes the HPTLC-MALDI MS analytical throughput both for lipids extracted in organic and aqueous phase. This study indicates that alkaline hydrolysis is necessary for the detection of low abundant species such as Gb3Cer and ceramides in serum and Gb4Cer, CerP and HexCer in muscle tissue. The high hydrophobicity of ceramides has been overcome by the development of HPTLC plate in chloroform:methanol/50:3.5, which increases the number and the intensity of low abundant Cer species. MS/MS analysis has been conducted directly on HPTLC plate allowing the molecular recognition; furthermore a dataset of spectra was acquired to create a database for future profiling of these molecules.

Sphingolipids in Obesity and Correlated Co-Morbidities: The Contribution of Gender, Age and Environment

“…Today, LC-ESI MS/MS is the most utilized method [41,42], but other on-line hyphenated techniques have been applied to sphingolipid research, like LC-NMR [43], CE-MS [44] and TLC/HPTLC-MS [45,46,47,48].…”

Analysis of Carbohydrates and Glycoconjugates by Matrix‐assisted Laser Desorption/Ionization Mass Spectrometry: An Update for 2015–2016

“…An HP‐TLC‐MALDI‐TOF/TOF MS method has been developed to profile sphingolipids and glycosphingolipids in muscle, brain, and serum (Torretta et al, 2016). Protocols for lipid extraction were established based on the characteristics of the tissue or/and fluids from which the target molecules were extracted.…”

Recent Advances in Combinations of TLC With MALDI and Other Desorption/Ionization Mass-Spectrometry Techniques

“…The additional MS data allowed to suggest that the colon epithelial cells synthesize globotriaosylceramide species with dihydroxylated sphinganine (d18:0), detected at m/z 1048.66 (d18:0, C16:0), and trihydroxylated sphinganine (t18:0) at m/z 1064.66 (t18:0, C16:0). Torretta et al (2016) have used HPTLC-MALDI-MS for the investigation of sphingolipid and glycosphingolipid profiles in the muscle, brain, and serum for creating a database of molecules for preclinical and clinical investigations. Based on the properties of the studied tissues and fluids, the specific protocols for lipid extraction were used by the authors to maximize the HPTLC-MALDI-MS analytical throughput both for lipids extracted in the organic and aqueous phases.…”