title: 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.
authors.affiliation: Department of Biomedical Sciences for Health, University of Milan, Segrate, Milan, Italy.
authors.forename: Enrica
authors.initials: E
authors.lastname: Torretta
authors.forename: Michele
authors.initials: M
authors.lastname: Vasso
authors.forename: Chiara
authors.initials: C
authors.lastname: Fania
authors.forename: Daniele
authors.initials: D
authors.lastname: Capitanio
authors.forename: Sonia
authors.initials: S
authors.lastname: Bergante
authors.forename: Marco
authors.initials: M
authors.lastname: Piccoli
authors.forename: Guido
authors.initials: G
authors.lastname: Tettamanti
authors.forename: Luigi
authors.initials: L
authors.lastname: Anastasia
authors.forename: Cecilia
authors.initials: C
authors.lastname: Gelfi
mesh_terms: D020384:Acidic Glycosphingolipids / Q000032:analysis* / Q000378:metabolism; D000818:Animals; D019540:Area Under Curve; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D002855:Chromatography, Thin Layer / Q000379:methods*; D016014:Linear Models; D051379:Mice; D032446:Myoblasts; D009439:Neuraminidase / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
chemical_list: D020384:Acidic Glycosphingolipids; C511796:Neu3 protein, mouse; D009439:Neuraminidase
keywords: Gangliosides; Glycosphingolipids; HPTLC-MALDI; Lipid analysis; MS