HIGH-SENSITIVITY MASS-SPECTRAL CHARACTERIZATION OF GLYCOSPHINGOLIPIDSFROM BOVINE ERYTHROCYTES, MOUSE KIDNEY AND FETAL CALF BRAIN

Stage-specific embryonic antigen (SSEA) glycosphingolipids (GSLs) foun d in the central nervous system are implicated in regulating cell-cell recognition, targeting and migration of cells during development. Thr ough the action of fucosyltransferase enzymes, SSEA-1 (Lewis(x)) glyco lipids are biosynthesized in the brain by fucosylation of lipid substr ates with the neolacto series glycolipid core structure [Gal beta 1 -- > 4GlcNAc beta 1 --> 3Gal beta 1 --> 4Glc beta 1 --> 1'Cer] (originall y termed paragloboside) or its higher analogs. In order to optimize me thodology for high sensitivity structural determinations of SSEA-1 typ e glycolipids from fetal calf brain, potential precursors and SSEA-1 g lycolipids of previously established structure were first isolated fro m bovine erythrocytes and beige mutant mouse kidney, purified by colum n chromatography and characterized by matrix-assisted laser desorption /ionisation time-of-flight (MALDI-TOF) MS, liquid secondary ionization mass spectrometry (LSIMS), and tandem mass spectrometry (MS/MS), amon g other techniques. Peracetylated derivatives were detected at the low femtomole level by MALDI-TOF MS and the subnanomole level by LSIMS. M ALDI-TOF MS produced mainly [M + Na](+) and [M + K](+) species. On the basis of the direct and tandem mass spectral analyses of peracetylate d and permethylated derivatives, the carbohydrate sequences in the sel ected bovine erythrocyte and mouse kidney GSL fractions were found to be consistent with those of glycolipids previously-reported from large r-scale studies of these sources. Their heterogeneous ceramide moietie s were characterized by collision induced decomposition (CID) MS/MS of abundant Z(0)-type ions in the LSI mass spectra of the permethylated GSLs. MALDI-PSD-TOF mass spectral analyses of low and subpicomole amou nts of derivatized GSL fractions from fetal calf brain provided carboh ydrate sequence information that indicates the presence of mono-and di fucosylated SSEA-1 neolacto series glycolipids and their nonfucosylate d analogs. (C) 1997 Elsevier Science B.V.