title: Quantitative GSL-glycome analysis of human whole serum based on an EGCase digestion and glycoblotting method.
abstract: Glycosphingolipids (GSLs) are lipid molecules linked to carbohydrate units that form the plasma membrane lipid raft, which is clustered with sphingolipids, sterols, and specific proteins, and thereby contributes to membrane physical properties and specific recognition sites for various biological events. These bioactive GSL molecules consequently affect the pathophysiology and pathogenesis 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 human serum based on endoglycoceramidase digestion and glycoblotting purification. We established two sample preparation protocols, one with and the other without GSL extraction 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 μl 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 μM. We further applied this method to TLC-prefractionated serum samples. These findings will assist the discovery of disease-related biomarkers by serum GSL-glycomics.
authors.affiliation: Laboratories of Medical and Functional Glycomics Graduate School of Advanced Life Science, Hokkaido University, Sapporo 001-0021, Japan yshinohara@sci.hokudai.ac.jp jfuru@sci.hokudai.ac.jp.
authors.forename: Jun-ichi
authors.initials: J
authors.lastname: Furukawa
authors.affiliation: Biomembrane and Biofunctional Chemistry, Graduate School of Advanced Life Science, Hokkaido University, Sapporo 001-0021, Japan.
authors.forename: Shota
authors.initials: S
authors.lastname: Sakai
authors.affiliation: Laboratories of Medical and Functional Glycomics Graduate School of Advanced Life Science, Hokkaido University, Sapporo 001-0021, Japan.
authors.forename: Ikuko
authors.initials: I
authors.lastname: Yokota
authors.affiliation: Laboratories of Medical and Functional Glycomics Graduate School of Advanced Life Science, Hokkaido University, Sapporo 001-0021, Japan.
authors.forename: Kazue
authors.initials: K
authors.lastname: Okada
authors.affiliation: Biomembrane and Biofunctional Chemistry, Graduate School of Advanced Life Science, Hokkaido University, Sapporo 001-0021, Japan.
authors.forename: Hisatoshi
authors.initials: H
authors.lastname: Hanamatsu
authors.affiliation: Shionogi Innovation Center for Drug Discovery, Shionogi & Co., Ltd., Sapporo 001-0021, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Kobayashi
authors.affiliation: Shionogi Innovation Center for Drug Discovery, Shionogi & Co., Ltd., Sapporo 001-0021, Japan.
authors.forename: Yasunobu
authors.initials: Y
authors.lastname: Yoshida
authors.affiliation: Shionogi Innovation Center for Drug Discovery, Shionogi & Co., Ltd., Sapporo 001-0021, Japan.
authors.forename: Kenichi
authors.initials: K
authors.lastname: Higashino
authors.affiliation: Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Sapporo 062-8517, Japan.
authors.forename: Tomohiro
authors.initials: T
authors.lastname: Tamura
authors.affiliation: Biomembrane and Biofunctional Chemistry, Graduate School of Advanced Life Science, Hokkaido University, Sapporo 001-0021, Japan.
authors.forename: Yasuyuki
authors.initials: Y
authors.lastname: Igarashi
authors.affiliation: Laboratories of Medical and Functional Glycomics Graduate School of Advanced Life Science, Hokkaido University, Sapporo 001-0021, Japan yshinohara@sci.hokudai.ac.jp jfuru@sci.hokudai.ac.jp.
authors.forename: Yasuro
authors.initials: Y
authors.lastname: Shinohara
mesh_terms: D002855:Chromatography, Thin Layer; D006026:Glycoside Hydrolases / Q000378:metabolism*; D006028:Glycosphingolipids / Q000097:blood* / Q000378:metabolism; D006801:Humans; D011134:Polysaccharides / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D006028:Glycosphingolipids; D011134:Polysaccharides; D006026:Glycoside Hydrolases; C050427:endoglycoceramidase
keywords: biomarker; blood; diagnostic tools; endoglycoceramidase; gangliosides; glycolipids; glycomics; glycosphingolipid; lipidomics; mass spectrometry; thin-layer chromatography