Advances on the compositional analysis of glycosphingolipids combining thin-layer chromatography with mass spectrometry

Abstract

Glycosphingolipids (GSLs), composed of a hydrophilic carbohydrate chain and a lipophilic ceramide anchor, play pivotal roles in countless biological processes, including infectious diseases and the development of cancer. Knowledge of the number and sequence of monosaccharides and their anomeric configuration and linkage type, which make up the principal items of the glyco code of biologically active carbohydrate chains, is essential for exploring the function of GSLs. As part of the investigation of the vertebrate glycome, GSL analysis is undergoing rapid expansion owing to the application of novel biochemical and biophysical technologies. Mass spectrometry (MS) takes part in the network of collaborations to further unravel structural and functional aspects within the fascinating world of GSLs with the ultimate aim to better define their role in human health and disease. However, a single-method analytical MS technique without supporting tools is limited yielding only partial structural information. Because of its superior resolving power, robustness, and easy handling, high-performance thin-layer chromatography (TLC) is widely used as an invaluable tool in GSL analysis. The intention of this review is to give an insight into current advances obtained by coupling supplementary techniques such as TLC and mass spectrometry. A retrospective view of the development of this concept and the recent improvements by merging (1) TLC separation of GSLs, (2) their detection with oligosaccharide-specific proteins, and (3) in situ MS analysis of protein-detected GSLs directly on the TLC plate, are provided. The procedure works on a nanogram scale and was successfully applied to the identification of cancer-associated GSLs in several types of human tumors. The combination of these two supplementary techniques opens new doors by delivering specific structural information of trace quantities of GSLs with only limited investment in sample preparation.

Shiga toxin glycosphingolipid receptors of Vero-B4 kidney epithelial cells and their membrane microdomain lipid environment

“…GSLs belong to the structurally very diverse group of sphingolipids (40)(41)(42) and have long been considered mysterious and puzzling riddles, beginning with the initial naming of "sphingosin" in the 1880s by the German physician and biochemist J. L. W. Thudichum due to their enigmatic ("Sphinx-like") properties ( 43,44 ). Their biosynthesis is rather complex ( 42 ) and they play important roles in the formation of the nervous system, where they are especially abundant (45)(46)(47).…”

Surface analysis of lipids by mass spectrometry: More than just imaging

“…By separating lipid classes prior to analysis the suppression effects of PC/SM are eliminated and low abundance lipid classes (such as PI) can be observed even from complex samples [81]. TLC/MALDI has shown much promise for the analysis of both phospholipids (PLs) and glycosphingolipids (GSLs) from extracts obtained from a diverse range of sources [79,[81][82][83][84][85]. Nevertheless it may be affected by: (i) incomplete desorption of lipids from the plate due to the high affinity of some lipids for 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 13 silica; (ii) poor penetration of the UV-laser beam into the silica layer such that only lipids close to the surface are removed; and (iii) the possibility of ablating and contaminating the instrument with silica.…”

Shiga toxin glycosphingolipid receptors in microvascular and macrovascular endothelial cells: differential association with membrane lipid raft microdomains

“…GSLs are built up from a hydrophobic ceramide moiety and a hydrophilic oligosaccharide residue ( 14,15 ). They are embedded with the ceramide moiety in the outer leaflet of the plasma membrane of animal cells, where they are clustered in a more ordered state within the generally disordered milieu of the membrane.…”

“…The localization of GSLs in the exofacial leafl et of the plasma membrane makes them excellent candidates as recognition structures in cell-cell interactions ( 16 ) and as receptors for numerous pathogens, such as viruses ( 17,18 ) and bacteria ( 19,20 ), as well as for bacterial toxins, including Shiga toxins (Stxs) ( 21,22 ). Much effort is currently spent employing (glyco)sphingolipidomics (23)(24)(25)(26) by use of various mass spectrometry technologies ( 15,(27)(28)(29)(30)(31) to better understanding their nature and functional role ( 32 ).…”

“…We report here on remarkable differences in microdomain composition with respect to the occurrence of Stx receptors and, moreover, on differential lipid raft stability toward cholesterol-depletion of the two endothelial cell types, where raft disintegration was found to be accompanied by loss of Stx receptors in DRM fractions. The newly developed technique combining thin-layer chromatography (TLC) separation and immunodetection of Stx receptors on the TLC plate with infrared matrix-assisted laser desorption/ionization orthogonal time-of-fl ight mass spectrometry (IR-MALDI-o-TOF-MS) served as an indispensable tool for structural analysis of small GSL amounts directly on the TLC plate ( 15 ).…”