title: 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.
authors.affiliation: Department of Biomedical Sciences for Health, University of Milan, Milan, Italy.|IRCCS Policlinico San Donato, Piazza Edmondo Malan, San Donato Milanese, Milan, Italy.
authors.forename: Enrica
authors.initials: E
authors.lastname: Torretta
authors.affiliation: IRCCS Policlinico San Donato, Piazza Edmondo Malan, San Donato Milanese, Milan, Italy.
authors.forename: Chiara
authors.initials: C
authors.lastname: Fania
authors.affiliation: Institute of Molecular Bioimaging and Physiology (IBFM), CNR, Milan, Italy.
authors.forename: Michele
authors.initials: M
authors.lastname: Vasso
authors.affiliation: Department of Biomedical Sciences for Health, University of Milan, Milan, Italy.|IRCCS Policlinico San Donato, Piazza Edmondo Malan, San Donato Milanese, Milan, Italy.
authors.forename: Cecilia
authors.initials: C
authors.lastname: Gelfi
mesh_terms: D000818:Animals; D015415:Biomarkers / Q000097:blood / Q000378:metabolism*; D001921:Brain / Q000378:metabolism; D002855:Chromatography, Thin Layer / Q000379:methods*; D006028:Glycosphingolipids / Q000097:blood / Q000378:metabolism*; D006868:Hydrolysis; D051379:Mice; D008810:Mice, Inbred C57BL; D018482:Muscle, Skeletal / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
chemical_list: D015415:Biomarkers; D006028:Glycosphingolipids
keywords: Gangliosides; Glycosphingolipids; HPTLC-MALDI MS; Multiplexing; Sphingolipids