Blood group A-active glycosphingolipids analysis by the combination of TLC-immunostaining assay and TLC/SIMS mass spectrometry.
Blood group A-active glycosphingolipids from human erythrocyte membranes were identified by the combination of thin-layer chromatography and matrix-assisted secondary ion mass spectrometry (TLC/SIMS). Partially purified lipid extracts were chromatographed by TLC and then blood group A-active glycolipids were detected by TLC-immunostaining assay using anti-A antibody. The parts of the plates which contained the same Rf area as anti-A positive spots were cut out and subjected to direct SIMS analysis. The TLC/SIMS spectra were quite similar to those obtained by ordinary SIMS. Detailed information, such as molecular weight, molecular species, ceramide portion, and oligosaccharide sequence, was obtained. Also, peracetylated blood group A-active glycolipids were analyzed in a similar manner. After the position of A-active glycolipids on a TLC plate was confirmed by in situ deacetylation and TLC-immunostaining, acetylated A-active glycolipids were also analyzed by the TLC/SIMS. Enhanced sensitivity was obtained with peracetylated glycolipids. Consequently, small amounts of unpurified bioactive glycolipids can be readily analyzed by TLC/SIMS.
Department of Biochemistry, Faculty of Medicine, Tokyo Medical and Dental University.
Y
Y
Kushi
K
K
Ogura
C
C
Rokukawa
S
S
Handa
D002240:Carbohydrate Sequence; D002855:Chromatography, Thin Layer / Q000379:methods; D004910:Erythrocyte Membrane / Q000032:analysis; D006028:Glycosphingolipids / Q000032:analysis* / Q000097:blood*; D006801:Humans; D007118:Immunoassay / Q000379:methods; D013058:Mass Spectrometry / Q000379:methods; D008969:Molecular Sequence Data
D006028:Glycosphingolipids