Glycoblotting-assisted O-glycomics: ammonium carbamate allows for highly efficient o-glycan release from glycoproteins.
ABSTRACT Glycoblotting, high throughput method for N-glycan enrichment analysis based on the specific chemical ligation between aminooxy/hydrazide-polymers/solids and reducing N-glycans released from whole serum and cellular glycoproteins, was proved to be feasible for selective enrichment analysis of O-glycans of common (mucin) glycoproteins. We established a standard protocol of glycoblotting-based O-glycomics in combination with nonenzymatic chemical treatment to release reducing O-glycans predominantly from various glycoprotein samples. It was demonstrated that the nonreductive condition employing a simple ammonium salt, ammonium carbamate, made glycoblotting-based enrichment analysis of O-glycans possible without significant loss or unfavorable side reactions. A general workflow of glycoblotting using a hydrazide bead (BlotGlyco H), on-bead chemical manipulations, and subsequent mass spectrometry allowed for rapid O-glycomics of human milk osteopontin (OPN) and urinary MUC1 glycoproteins purified from healthy donors in a quantitative manner. It was revealed that structures of O-glycans in human milk OPN were varied with habitual fucosylation and N-acetyllactosamine units. It was also suggested that purified human urinary MUC1 was modified preferentially by sialylated O-glycans (94% of total) with 7:3 ratio of core 1 to core 2 type O-glycans. Versatility of the present strategy is evident because this method was proved to be suited for the enrichment analysis of general biological and clinical samples such as human serum and urine, cultured human cancer cells, and formalin-fixed paraffin-embedded tissue sections. It is our belief that the present protocols would greatly accelerate discovery of disease-relevant O-glycans as potential biomarkers.
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Article: Glycoprotein synthesis: an update.Chemical Reviews 01/2009; 109(1):131-63. · 41.30 Impact Factor
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ABSTRACT: The endoplasmic reticulum (ER) is the site of folding for proteins that are resident in the ER or that are destined for the Golgi, endosomes, lysosomes, the plasma membrane, or secretion. Cotranslational addition of preassembled glucose(3)-mannose(9)-N-acetylglucosamine(2) core oligosaccharides (N-glycosylation) is a common event for polypeptides synthesized in this compartment. Protein-bound oligosaccharides are exposed to several ER glycanases that sequentially remove terminal glucose or mannose residues. Their activity must be tightly regulated because the N-glycan composition determines whether the associated protein is subjected to folding attempts in the ER lumen or whether it is retrotranslocated into the cytosol and degraded.Nature Chemical Biology 07/2007; 3(6):313-20. · 12.95 Impact Factor