Characterization of novel myelin components 3‐O‐acetyl‐sphingosine galactosylceramides by electrospray ionization Q‐TOF MS and MS/CID‐MS of Li+ adducts

Abstract

Glycosphingolipids with R(f) values higher than those of monoglycosylceramides (MGCs) in normal phase HPTLC appear to be normal components of myelin. A series of such low polarity components, referred to as 'fast moving cerebrosides' (FMCs), have been isolated from rat brain, and two of these fractions (FMC-1 and FMC-2) were found to be novel derivatives of galactosylceramide (GalCer) exhibiting O-acetylation at the 3-hydroxy group of the sphingoid moiety, and incorporating either non-hydroxy or 2-hydroxy fatty-N-acylation (Dasgupta S, Levery SB, Hogan EL. J. Lipid Res. 2002; 43: 751-761). Similar to the parent compounds, the 3-O-acetyl-sphingoid derivatives exhibit considerable diversity with respect to fatty-N-acyl chain length, manifested by heterogeneous molecular ion (Li(+) adduct) profiles. However, a detailed analysis of the individual molecular variants ('lipoforms'), e.g. by tandem MS/CID-MS analysis, was not carried out. In addition, several other FMCs distinguished by even lower polarity (higher HPTLC R(f) values) were isolated but have remained uncharacterized. For this study, analysis of both the known and unknown FMC components was carried out by positive ion ESI-MS and MS/CID-MS of their Li(+) adducts on a Q-TOF mass spectrometer. Since a Q-TOF instrument has not yet been applied to MS of lithiated cerebrosides and FMCs, MS/CID-MS spectra of bovine brain GalCer (both types) and the previously characterized rat brain FMCs (FMC-1 and FMC-2), having 3-O-acetylation of the sphingoid, were systematically acquired and their fragmentation behavior compared. This was followed by systematic analysis of previously uncharacterized FMC fractions (FMC-3 through FMC-5/6/7). The GalCer and FMC components proved to be amenable to analysis by this technique, and the data confirm that the latter are all related 3-O-acetyl-sphingoid derivatives, with the higher R(f) components carrying additional O-acetyl modifications on the galactosyl residue, which further reduce their polarity. The utility of the technique, the structures of unknown FMCs, and their characteristic fragmentation patterns are described.