A New Strategy for Identification of N-Glycosylated Proteins and Unambiguous Assignment of Their Glycosylation Sites Using HILIC Enrichment and Partial Deglycosylation

Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark.
Journal of Proteome Research (Impact Factor: 4.25). 06/2004; 3(3):556-66. DOI: 10.1021/pr034112b
Source: PubMed


Characterization of glycoproteins using mass spectrometry ranges from determination of carbohydrate-protein linkages to the full characterization of all glycan structures attached to each glycosylation site. In a novel approach to identify N-glycosylation sites in complex biological samples, we performed an enrichment of glycosylated peptides through hydrophilic interaction liquid chromatography (HILIC) followed by partial deglycosylation using a combination of endo-beta-N-acetylglucosaminidases (EC After hydrolysis with these enzymes, a single N-acetylglucosamine (GlcNAc) residue remains linked to the asparagine residue. The removal of the major part of the glycan simplifies the MS/MS fragment ion spectra of glycopeptides, while the remaining GlcNAc residue enables unambiguous assignment of the glycosylation site together with the amino acid sequence. We first tested our approach on a mixture of known glycoproteins, and subsequently the method was applied to samples of human plasma obtained by lectin chromatography followed by 1D gel-electrophoresis for determination of 62 glycosylation sites in 37 glycoproteins.

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    • "before subsequent LC - MS / MS to identify glycosyla - tion sites ( Parker et al . , 2011 ; Qu et al . , 2011 ; Kuo et al . , 2012 ) . The HILIC approach can also be applied to identification of core fucosylated N - glycans and O - glycosylation site mapping of glycoproteins through partial deglycosylation performed by a specific enzyme reaction ( Hagglund et al . , 2004 , 2007 ) . In these studies , digestion of glycopeptides is conducted by using endo - beta - N - acetylglucosaminidases ( Endo H ) that cleave the glyco - sidic bond between the two GlcNAc residues in the conserved N - glycan core structure . Thereby , single GlcNAc residues with putative fucosyl side moieties are leaved onto the peptid"
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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 · 7.71 Impact Factor
    • "To encompass these limitations and also to enrich low-abundance glycoproteins/glycopeptides from biofluids or tissues, different analytical strategies have been described and involve, for example, affinity capture by lectins [29] or hydrophilic interaction chromatography (HILIC). [30] [31] These two approaches, as opposed to hydrazide-based glycopeptide enrichment, [32] keep the glycan moieties of the glycopeptides intact upon enrichment thus rendering them amenable for further site-specific analysis. The situation is even more challenging when considering the difficulty of interpreting the tandem mass (MS/MS) spectra of intact glycopeptides (especially those obtained under collision-induced dissociation conditions) for obtaining site-specific information and the lack of efficient software(s) for doing so. "
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    ABSTRACT: RationaleGlycosylation is one of the most complex types of post-translational modifications of proteins. The alteration of glycans bound to proteins from cerebrospinal fluid (CSF) in relation to disorders of the central nervous system is a highly relevant subject, but only few studies have focused on the glycosylation of CSF proteins.Methods Reproducible profiles of CSF N-glycans were first obtained by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry after permethylation. Tryptic glycopeptides from CSF proteins were also enriched by hydrophilic interaction, and the resulting extracts divided into two equal aliquots. A first aliquot was enzymatically deglycosylated and analyzed by nano-liquid chromatography/tandem mass spectrometry while the second one, containing intact enriched glycopeptides, was directly analyzed. Site-specific data were obtained by combining the data from these three experiments.ResultsWe describe the development of a versatile approach for obtaining site-specific information on the N-glycosylation of CSF glycoproteins. Under these conditions, 124 N-glycopeptides representing 55 N-glycosites from 36 glycoproteins were tentatively identified. Special emphasis was placed on the analysis of glycoproteins/glycopeptides bearing 'brain-type' N-glycans, representing potential biologically relevant structures in the field of neurodegenerative disorders. Using our workflow, only a few proteins were shown to carry such particular glycan motifs.Conclusions We developed an approach combining N-glycomics and N-glycoproteomics and underline its usefulness to study the site-specific glycosylation of major human CSF proteins. The final rather long-term objective is to combine these data with those from other omics approaches to delve deeper into the understanding of particular neurological disorders. Copyright © 2015 John Wiley & Sons, Ltd.
    Rapid Communications in Mass Spectrometry 03/2015; 29(6). DOI:10.1002/rcm.7125 · 2.25 Impact Factor
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    • "Glycoproteins can be enriched by using sugar-speci fi c antibody lectins (Gabius et al. 2002 ; Yang and Hancock 2004 ) and TiO 2 columns (Larsen et al. 2007 ) . Glycosidase D/H treatment and MS/MS facilitated protein identi fi cation and assignment of glycosylation sites (Hagglund et al. 2004 ) . Acetylated peptides can be enriched by using resin-coupled antibodies to acetyllysine (Kim et al. 2006 ) . "

    Proteomics in Foods, Edited by Fidel Toldrá, Leo M. L. Nollet, 01/2013: chapter 7: pages 111-125; Springer US., ISBN: 9781461456254
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