Quantitative GSL-glycome analysis of human whole serum based on an EGCase digestion and glycoblotting method

Glycosphingolipids (GSLs) are lipid molecules linked to carbohydrate units that form the plasma mem- brane lipid raft, which is clustered with sphingolipids, ste- rols, and specifi c proteins, and thereby contributes to membrane physical properties and specifi c recognition sites for various biological events . These bioactive GSL mole- cules consequently affect the pathophysiology and patho- genesis of various diseases. Thus, altered expression of GSLs in various diseases may be of importance for disease- related biomarker discovery. However, analysis of GSLs in blood is particularly challenging because GSLs are present at extremely low concentrations in serum/plasma. In this study, we established absolute GSL-glycan analysis of hu- man serum based on endoglycoceramidase digestion and glycoblotting purifi cation. We established two sample prep- aration protocols, one with and the other without GSL ex- traction using chloroform/methanol. Similar amounts of GSL-glycans were recovered with the two protocols. Both protocols permitted absolute quantitation of GSL-glycans using as little as 20 l of serum. Using 10 healthy human serum samples, up to 42 signals corresponding to GSL- glycan compositions could be quantitatively detected, and the total serum GSL-glycan concentration was calculated to be 12.1-21.4 M. We further applied this method to TLC- prefractionated serum samples. These fi ndings will assist the discovery of disease-related biomarkers by serum GSL- glycomics. —Furukawa, J-i., S. Sakai, I. Yokota, K. Okada, H. Hanamatsu, T. Kobayashi, Y. Yoshida, K. Higashino, T. Tamura, Y. Igarashi, and Y. Shinohara. Quantitative GSL- glycome analysis of human whole serum based on an EGCase digestion and glycoblotting method. J. Lipid Res. 2015. 56: 2399-2407.