Solid‐Phase Oligosaccharide Tagging (SPOT): Validation on Glycolipid‐Derived Structures

Abstract

Glycosylation is the most abundant form of post-translational modification of proteins. Over 50 % of all protein sequences are glycosylated in eukaryotic systems.[1] Glycosylated lipids are also widespread in animal cells: they constitute up to 5-10 % of the cell membrane content and are responsible for a wide array of pathological disorders.[2]The structural analysis, or sequencing, of the oligosaccharide chains of glycoproteins and glycolipids is much more complex than that of DNA or proteins, as the molecules are generally branched and the linkage between sugar residues introduces a new a or b stereocenter. The major analytical approaches [3] rely on the detection of oligosaccharides either by mass spectrometry (MS) or pulsed amperometry. Oligosaccharides released from glycoproteins or glycolipids by chemical or enzymatic methods are also frequently labeled at the reducing end to afford increased sensitivity of detection. [4]

BlotGlycoABC™, an Integrated Glycoblotting Technique for Rapid and Large Scale Clinical Glycomics

“…The solution was removed by applying vacuum, and then the beads were washed with ACN, water, and MeOH (3 ϫ 300 l) successively. The hydrazone linkage between the oligosaccharide and the fluorescent probe on the bead was stabilized by incubation with a reducing reagent, 0.8 M borane-pyridine, according to the method reported previously (16). The reduction was performed at room temperature for 30 min in the dark.…”

“…The hydrazide group reacts with aldehyde or ketone groups that are very rare in common biological samples except carbohydrates having a reducing hemiacetal terminus. Formation of the hydrazone bond between the BlotGlycoABC bead and glycans is reversible so that parental reducing sugars can be released; otherwise it could be reduced for conversion to a stable C-N bond (16). This chemistry is suitable to enrich carbohydrates from complex biological materials even in the presence of various amines and reagents used in proteomics sample preparation that contain a variety of reagents such as DTT, iodoacetamide, detergents, etc.…”

“…Our recent efforts have been directed to the establishment of practical methods for glycan enrichment, namely "glycoblotting" (15)(16)(17)(18)(19)(20). The optimized protocol requires only 5 l of human serum for the quantitative profiling of 30 -40 kinds of major glycoforms within 5-8 h (19).…”

BlotGlyco and glycoblotting for large scale, high throughput glycomics

“…Unlike the DNA and proteins, glycans are not generally ampliˆed in vitro. Thus, it is important to retrieve su‹cient amounts of glycans without any bias (1). The new technology of glycoblotting (using BlotGlyco bead) that we have developed (2,3) allows enrichment of glycans from a complex samples.…”

Synthesis and Application of Glycopeptide and Glycoprotein Mimetics

“…In order to fix the sugar in a defined acyclic form, subsequent reduction with, e.g., sodium cyanoborohydride to give the corresponding hydroxylamine and hydrazide, respectively, may be carried out. This process, however, generates new nucleophilic centers (which might have additional utility [148]) and is no longer a sheer ligation reaction.…”