Glycoblotting-Assisted O-Glycomics: Ammonium Carbamate Allows for Highly Efficient O-Glycan Release from Glycoproteins

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Analytical Chemistry (Impact Factor: 5.64). 11/2010; 82(24):10021-9. DOI: 10.1021/ac101599p
Source: PubMed


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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    • "zed . Unfortunately this glycan - targeting approach has disadvantage such that information for glycoprotein or glycosyl - ation site generating a specific glycan component of interest disappears inevitably during glycan - preparing process via deglycosylation reaction by enzymatic or chemical method ( Maley et al . , 1989 ; Wells et al . , 2002 ; Miura et al . , 2010 ) . As a supplementary approach for the glycan - targeting method , the analysis of the deglycosylated protein parts of glycoproteins that drop glycan moieties via deglycosylation , is useful for glycopro - tein identification and glycosite ( lately , deglycosylated ) identifi - cation through profiling experiment by LC / tandem MS . Th"
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    Mass Spectrometry Reviews 04/2015; 34(2). DOI:10.1002/mas.21428 · 7.71 Impact Factor
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    • "Improved yield has been reported with both bovine fetuin and porcine stomach mucin compared with the classical strong base method [27]. Highly efficient O-glycan release has been recently demonstrated with ammonium carbamate [29]. However, quantitative aspects have not been addressed. "
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    Analytical Biochemistry 10/2013; 446(1). DOI:10.1016/j.ab.2013.10.019 · 2.22 Impact Factor
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    • "After obtaining the mucin samples from B6, CBA, and BALB mice, the O-glycomic profiles of the three mouse strains were compared . O-glycans were liberated from the mucin samples using ammonium carbamate, and then purified and labeled with aoWR tag using the glycoblotting technique [13] [14] [15]. After the removal of excess reagent, the labeled glycans were analyzed by MALDI- TOF MS. "
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