US EPA

3721883 

Journal Article 

Glycoblotting-assisted O-glycomics: ammonium carbamate allows for highly efficient o-glycan release from glycoproteins 

Miura, Y; Kato, K; Takegawa, Y; Kurogochi, M; Furukawa, J; Shinohara, Y; Nagahori, N; Amano, M; Hinou, H; Nishimura, S 

2010 

Yes 

Analytical Chemistry
ISSN: 0003-2700
EISSN: 1520-6882 

82 

24 

10021-10029 

English 

21077635 

10.1021/ac101599p 

WOS:000285215800009 

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. 

Ammonium carbamates; Ammonium salt; Chemical ligation; Chemical manipulation; Chemical treatments; Clinical samples; Formalin; Fucosylation; Glycoblotting; Glycomics; Glycoprotein samples; High-throughput method; Human cancer cells; Human milk; Human serum; MUC1 glycoprotein; N-glycan; N-Glycans; Non-enzymatic; O-Glycans; Osteopontin; Selective enrichment; Side reactions; Standard protocols; Tissue sections; Ammonium compounds; Body fluids; Formaldehyde; Glycoproteins; Mass spectrometry; Paraffin waxes; Paraffins; Purification; Chemical analysis; biological marker; carbamic acid; carbamic acid derivative; glycoprotein; polysaccharide; article; chemistry; glycobiology; human; methodology; Biological Markers; Carbamates; Glycomics; Glycoproteins; Humans; Polysaccharides