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
Source: PubMed

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.

1 Follower
15 Reads
  • Source
    • "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"
    [Show abstract] [Hide abstract]
    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 · 7.71 Impact Factor
  • Source
    • "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. "
    [Show abstract] [Hide abstract]
    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 · 2.30 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: As drastic structural changes in cell-surface glycans of glycoproteins and glycosphingolipids, as well as serum glycoproteins, are often observed during cell differentiation and cancer progression, it is considered that glycans can be potential candidates for novel diagnostic and therapeutic biomarkers. Although there have been substantial advances in our understanding of the effects of glycosylation on some biological systems, we still do not fully understand the significance and mechanism of glycoform alteration that is widely observed in many human diseases. This is due to the highly complicated structures of the glycans and the extremely tedious and time-consuming processes required for their separation from complex mixtures and their subsequent analysis. As a result, with a few notable exceptions, the therapeutic potential of complex glycans has not been well exploited. This article is focused on the state of the art and current advances in glycomics, and efforts for the development of automated glycan analysis, which should greatly accelerate functional glycobiology and its medical/pharmaceutical applications. The "glycoblotting method" is the only method currently available that allows rapid and large-scale clinical glycomics of human whole-serum glycoproteins, because it requires very little material and, when combined with an automated system "SweetBlot," takes only ∼14h to complete whole glycan profiling by mass spectrometry. The upcoming goal is to combine glycoblotting methods and various MS-based platforms for the development of a fully automated glycan analytical system and accelerating research to discover highly sensitive and clinically important biomarker molecules.
    Advances in Carbohydrate Chemistry and Biochemistry 01/2011; 65:219-71. DOI:10.1016/B978-0-12-385520-6.00005-4 · 2.10 Impact Factor
Show more