Glycoblotting-Assisted O-Glycomics: Ammonium Carbamate Allows for Highly Efficient O-Glycan Release from Glycoproteins
Ezose Sciences, Inc., 25 Riverside Drive Pine Brook, New Jersey 07058, United States. Analytical Chemistry
(Impact Factor: 5.64).
11/2010; 82(24):10021-9. DOI: 10.1021/ac101599p
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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Available from: Jong Shin Yoo
- "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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ABSTRACT: Mass spectrometry (MS) has been a core technology for high sensitive and high-throughput analysis of the enriched glycoproteome in aspects of quantitative assays as well as qualitative profiling of glycoproteins. Because it has been widely recognized that aberrant glycosylation in a glycoprotein may involve in progression of a certain disease, the development of efficient analysis tool for the aberrant glycoproteins is very important for deep understanding about pathological function of the glycoprotein and new biomarker development. This review first describes the protein glycosylation-targeting enrichment technologies mainly employing solid-phase extraction methods such as hydrizide-capturing, lectin-specific capturing, and affinity separation techniques based on porous graphitized carbon, hydrophilic interaction chromatography, or immobilized boronic acid. Second, MS-based quantitative analysis strategies coupled with the protein glycosylation-targeting enrichment technologies, by using a label-free MS, stable isotope-labeling, or targeted multiple reaction monitoring (MRM) MS, are summarized with recent published studies. © 2014 The Authors. Mass Spectrometry Reviews Published by Wiley Periodicals, Inc. Rapid Commun. Mass Spectrom.
Available from: Zoran Sosic
- "Improved yield has been reported with both bovine fetuin and porcine stomach mucin compared with the classical strong base method . Highly efficient O-glycan release has been recently demonstrated with ammonium carbamate . However, quantitative aspects have not been addressed. "
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ABSTRACT: The principal aim of this manuscript is to demonstrate the optimization and fine tuning of quantitative and non-selective analysis of O-linked glycans released from therapeutic glycoproteins. Two approaches for quantitative release of O-linked glycans have been examined. These include ammonia-based β-elimination and hydrazinolysis deglycosylation strategies. A significant discrepancy in deglycosylation activity has been observed between ammonia-based and hydrazinolysis procedures. Specifically, the release of O-glycans from glycoproteins has been about 20-30 times more efficient with hydrazine compared to ammonia-based β-elimination reagent. In addition, the ammonia-based reagent has demonstrated bias in the release of particular glycan species. A robust quantitative hydrazinolysis procedure has been developed for characterization of O-glycans. The method performance parameters have been evaluated. It has been shown that this procedure is superior for quantitative non-selective release of O-glycans. Identity confirmation and structure elucidation of O-glycans from HILIC fractions has also been demonstrated using Linear Ion Trap Fourier Transform Mass Spectrometry (LTQ FT MS) with the mass accuracy below 1 ppm.
Available from: Kentaro Kato
- "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   . After the removal of excess reagent, the labeled glycans were analyzed by MALDI- TOF MS. "
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ABSTRACT: Mouse strain-specific differences in the carbohydrate composition of intestinal mucins were hypothesized to account for strain-dependent susceptibility to Entamoeba histolytica. To test this hypothesis, intestinal mucins from susceptible and resistant inbred strains of mice were analyzed for their O-glycan content and for their ability to inhibit amoebic adherence to (GalNAc)12-27-HSA neo-glycoproteins. The results showed that the colorectal mucin O-glycan of susceptible CBA mice was lower in sialic acid content than that of resistant C57BL/6 and BALB/c mice. Mucins from CBA mice were more potent inhibitors of Entamoeba histolytica adherence to neo-glycoproteins than were mucins from C57BL/6 or BALB/c mice. Consistent with the role of terminal Gal/GalNAc as a receptor for amoebic adherence, sialidase treatment of C57BL/6 and BALB/c colorectal mucins increased their ability to inhibit E. histolytica adherence to the neo-glycoproteins. These results provide evidence of mouse strain-specific differences in the sialic acids content of mucin O-glycans. These dissimilarities likely contribute to the differential susceptibility of the three mouse strains to Entamoeba histolytica infection.
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