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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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.Mass Spectrometry Reviews 04/2015; 34(2). DOI:10.1002/mas.21428 · 8.05 Impact Factor
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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.Biochemical and Biophysical Research Communications 05/2013; 436(2). DOI:10.1016/j.bbrc.2013.05.085 · 2.28 Impact Factor
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ABSTRACT: A large set of glycome information was obtained from egg white proteins of 88 samples from Galloanserae (63 Anseriformes and 25 Galliformes). The data were obtained on whole N-glycan structures and types of sialic acids of these egg whites by glycoblotting-based high-throughput and quantitative glycomics. The results revealed clear trends and complexity patterns as well as diversity among taxonomic groups. It is well-known that chicken, a representative domesticated poultry involved in Galliformes, can become an influenza host. However, our data demonstrate that duck, wild goose, and swan of Anseriformes are representative migratory birds that are known as natural hosts of the influenza virus. Hierarchical clustering analysis of the expression pattern of N-glycome (total of 61 N-glycan peaks) revealed that the members of Galloanserae can be classified into two major groups and five submajor clusters (clusters 1-5) on the basis of simple m/z values obtained by MALDI-TOF MS. It is clear that expression patterns of N-glycomes in the five clusters are influenced significantly by the features such as the body size of the birds, rather than by the difference of the family. On the other hand, quantitative analysis showed that the total amounts of sialic acids in egg whites of Galliformes were distinctly larger than those of Anseriformes. However, it was also revealed in Anseriformes that Neu5Gc and KDN, in addition to common Neu5Ac, were expressed significantly in both N- and O-glycans of glycoproteins and glycosphingolipids, suggesting the influence of their lifestyles and diet. This is the first report that KDN exists in egg white. These results and the environmental factors are discussed preliminarily with respect to their evolutionary lineage.Biochemistry 05/2011; 50(21):4757-74. DOI:10.1021/bi101940x · 3.19 Impact Factor