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.

Keywords: biomarker; blood; diagnostic tools; endoglycoceramidase; gangliosides; glycolipids; glycomics; glycosphingolipid; lipidomics; mass spectrometry; thin-layer chromatography.

Fig. 1.

MALDI-TOF MS spectra showing quantitative human GSL-glycan profiles. C/M-extracted serum in the presence of EGCase (A) and C/M-extracted serum in the absence of EGCase (B) (protocol 1). Nonextracted serum in the presence of EGCase (C) and nonextracted serum in the absence of EGCase (D) (protocol 2). The asterisks shown in (C) and (D) indicate peptide signals. The double asterisks indicate N-glycan signals.

Fig. 2.

Streamlined protocol for the analysis of GSL-glycans obtained from human serum.

Fig. 3.

Ionization efficiency of various aoWR-labeled glycans in MALDI-TOF MS. Fourteen glycans labeled with aoWR were analyzed by MALDI-TOF MS in triplicate. The peak areas of the glycans relative to those of an internal standard (A2GN1) are plotted (mean ± SD). Equimolar amounts of glycans were prepared by acid hydrolysis and sequential enzymatic digestions of A2, A2F, and A2GN1. The prepared glycan structures are listed in supplementary Table 2.

Fig. 4.

Linear dynamic ranges of quantification of various GSL-glycans using protocol 1. The ion peak areas of GSL-glycans in MALDI-TOF MS were normalized to those of 20 pmol of A2GN1 as an internal standard.

Fig. 5.

Quantitative GSL-glycan analysis of healthy human serum. Both the absolute quantity of GSL-glycans (i.e., the size of the glycome) and the relative GSL-glycan composition are represented for each serum sample from healthy subjects. The areas of the circles represent the amount of total GSL-glycans per 100 μl of serum. The signal numbers correspond to those described in supplementary Table 4.

Fig. 6.

Biosynthesis pathway of Le^a/type I H antigen, Le^b, and ALe^b.