title: Evaluation of the photooxidation of galactosyl- and lactosylceramide by electrospray ionization mass spectrometry.
abstract: BACKGROUND
  Glycosphingolipids are important lipid molecules namely as constituents of the plasma membrane organized in lipid rafts, in signal transduction, and cell-cell communication. Although many human diseases are associated with oxidative stress and lipid oxidation, a link between oxidative stress and modification of glycosphingolipids has never been addressed.
  
  
  METHODS
  In this study, the structural changes caused by UVA-induced photooxidation of galactosyl- (GalCer) and lactosylceramide (LacCer) molecular species were studied by electrospray ionization mass spectrometry (ESI-MS and MS/MS), using a quadrupole time-of-flight (QTOF) mass spectrometer and high-performance liquid chromatography/tandem mass spectrometry with a C5 stationary phase (C5 HPLC/MS/MS) using a linear ion trap.
  
  
  RESULTS
  ESI-MS spectra of GalCer and LacCer after photooxidation showed new ions with a mass shift of +32 Da when compared with the ions of the non-modified glycosphingolipids. These new species were assigned as hydroperoxyl derivatives, confirmed by HPLC/MS/MS and through FOX 2 assay. In the ESI-MS and LC/MS of lactosylceramide a new ion with lower m/z value, assigned as glucosylceramide (GlcCer) + 32 Da, was also detected and proposed to be formed due to oxidative cleavage of lactosyl moieties. ESI-MS/MS of the oxidized species allowed us to infer the presence of isomeric hydroperoxyl derivatives, with the hydroperoxyl moiety either linked to the sphingosine backbone or in the unsaturated acyl chain. Oxidation in the sugar moieties was observed in the case of LacCer, suggesting an oxidation via radical reactive oxygen species that can induce the oxidative cleavage of the lactosyl moiety.
  
  
  CONCLUSIONS
  This study shows that glycosphingolipids are prone to oxidation and the identified mass spectrometry fingerprint of oxidized galactosyl- and lactosylceramide species will support their future identification in lipidomic studies of biological samples under oxidative conditions.
authors.affiliation: Mass Spectrometry Centre, UI-QOPNA, Department of Chemistry, University of Aveiro, 3810-193, Aveiro, Portugal.
authors.forename: Deolinda
authors.initials: D
authors.lastname: Santinha
authors.forename: Emanuel
authors.initials: E
authors.lastname: Ferreira-Fernandes
authors.forename: Tânia
authors.initials: T
authors.lastname: Melo
authors.forename: Eduarda M P
authors.initials: EM
authors.lastname: Silva
authors.forename: Elisabete
authors.initials: E
authors.lastname: Maciel
authors.forename: Margarida
authors.initials: M
authors.lastname: Fardilha
authors.forename: Pedro
authors.initials: P
authors.lastname: Domingues
authors.forename: M Rosário M
authors.initials: MR
authors.lastname: Domingues
mesh_terms: D015703:Antigens, CD / Q000737:chemistry* / Q000528:radiation effects; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D005699:Galactosylceramides / Q000737:chemistry* / Q000528:radiation effects; D007790:Lactosylceramides / Q000737:chemistry* / Q000528:radiation effects; D010084:Oxidation-Reduction; D055668:Photochemical Processes; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D053719:Tandem Mass Spectrometry; D014466:Ultraviolet Rays
chemical_list: D015703:Antigens, CD; D005699:Galactosylceramides; D007790:Lactosylceramides; C009744:CDw17 antigen