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J. Biochem, 1990, Vol. 107, No. 5 685-688
© 1990 Japanese Biochemical Society

research-article

Blood Group A-Active Glycosphingolipids Analysis by the Combination of TLC-Immunostaining Assay and TLC/SIMS Mass Spectrometry1

Yasunori Kushi, Kiyoshi Ogura, Chiaki Rokukawa and Shizuo Handa

Department of Biochemistry, Faculty of Medicine, Tokyo Medical and Dental University Bunkyo-ku, Tokyo 113

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 chromato-graphed by TLC and then blood group A-active glycolipids were detected by TLC-immuno-staining 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.

1 This study was supported in part by a Grant-in-Aid from the Ministry of Education, Science and Culture of Japan and a grant from the Tokyo Biochemical Society Foundation, and parts of this study were presented at the 60th Annual Meeting of the Japanese Biochemical Society (September, 1987, Kanazawa) and at the third Rinshoken Symposium (September, 1988, Tokyo).


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