Lectin affinity capture, isotope-coded tagging and mass spectrometry to identify N-linked glycoproteins (original) (raw)
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Journal of Proteome Research, 2004
Characterization of glycoproteins using mass spectrometry ranges from determination of carbohydrateprotein 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--N-acetylglucosaminidases (EC 3.2.1.96). 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.
Mapping human N-linked glycoproteins and glycosylation sites using mass spectrometry
TrAC Trends in Analytical Chemistry, 2019
N-linked glycoprotein is a highly interesting class of proteins for clinical and biological research. Over the last decade, large-scale profiling of N-linked glycoproteins and glycosylation sites from biological and clinical samples has been achieved through mass spectrometry-based glycoproteomic approaches. In this paper, we reviewed the human glycoproteomic profiles that have been reported in more than 80 individual studies, and mainly focused on the N-glycoproteins and glycosylation sites identified through their deglycosylated forms of glycosite-containing peptides. According to our analyses, more than 30,000 glycosite-containing peptides and 7,000 human glycoproteins have been identified from five different body fluids, twelve human tissues (or related cell lines), and four special cell types. As the glycoproteomic data is still missing for many organs and tissues, a systematical glycoproteomic analysis of various human tissues and body fluids using a uniform platform is still needed for an integrated map of human N-glycoproteomes.
Site-Specific Glycan-Peptide Analysis for Determination of N -Glycoproteome Heterogeneity
Journal of Proteome Research, 2013
A combined glycomics and glycoproteomics strategy was developed for the site-specific analysis of N-linked glycosylation heterogeneity from a complex mammalian protein mixture. Initially, global characterization of the N-glycome was performed using porous graphitized carbon liquid chromatography−tandem mass spectrometry (PGC-LC−MS/MS) and the data used to create an N-glycan modification database. In the next step, tryptic glycopeptides were enriched using zwitterionic hydrophilic interaction liquid chromatography (Zic-HILIC) and fractionated by reversed-phase liquid chromatography (RPLC; pH 7.9). The resulting fractions were each separated into two equal aliquots. The first set of aliquots were treated with peptide-N-glycosidase F (PNGase F) to remove Nglycans and the former N-glycopeptides analyzed by nano-RPLC-MS/MS (pH 2.7) and identified by Mascot database search. This enabled the creation of a glycopeptide-centric concatenated database for each fraction. The second set of aliquots was analyzed directly by nanoRPLC-MS/MS (pH 2.7), employing fragmentation by CID and HCD. The assignment of glycan compositions to peptide sequences was achieved by searching the N-glycopeptide HCD MS/MS spectra against the glycopeptide-centric concatenated databases employing the Nglycan modification database. CID spectra were used to assign glycan structures identified in the glycomic analysis to peptide sequences. This multidimensional approach allowed confident identification of 863 unique intact N-linked glycopeptides from 161 rat brain glycoproteins.
A lectin array-based methodology for the analysis of protein glycosylation
Journal of Biochemical and Biophysical Methods, 2007
Glycosylation is the most versatile and one of the most abundant protein modifications. It has a structural role as well as diverse functional roles in many specific biological functions, including cancer development, viral and bacterial infections, and autoimmunity. The diverse roles of glycosylation in biological processes are rapidly growing areas of research, however, Glycobiology research is limited by the lack of a technology for rapid analysis of glycan composition of glycoproteins. Currently used methods for glycoanalysis are complex, typically requiring high levels of expertise and days to provide answers, and are not readily available to all researcher.
NATO Science for Peace and Security Series A: Chemistry and Biology, 2008
Recently, we reported the characterization of the glycans attached at the 11 N-glycosylation sites of Hepatitis C virus E2 envelope glycoprotein by tandem mass spectrometry. Infections caused by Hepatitis C virus represent the main cause of liver diseases such as hepatitis, cirrhosis and hepatocellular carcinoma. The N-linked sugars consist primarily of high mannose glycans, with structures ranging from the minimal core structure, Man3GlcNAc2 (Man3) up to 12 hexose residues attached to the GlcNAc-ß(1-4)-GlcNAc core (depicted as Hex3Man9GlcNAc2). Furthermore, the site N41 (N423) was observed to contain complex type glycans with the structures Man3-GlcNAc and Man3-GlcNAcFuc, in addition to the high mannose population Man3 through Man6, while the site N48 (N430) was occupied exclusively with complex type glycans (Man3-Fuc, Man3-GlcNAcFuc and Man3-GlcNAc2Fuc). The present contribution summarizes our experimental observations upon the factors which may have an impact on the CID tandem mass spectra of glycopeptides.
Journal of proteome research, 2017
Protein glycosylation can have an enormous variety of biological consequences, reflecting the molecular diversity encoded in glycan structures. This same structural diversity has imposed major challenges on the development of methods to study the intact glycoproteome. We recently introduced a method termed isotope-targeted glycoproteomics (IsoTaG), which utilizes isotope recoding to characterize azidosugar-labeled glycopeptides bearing fully intact glycans. Here, we describe the broad application of the method to analyze glycoproteomes from a collection of tissue-diverse cell lines. The effort was enabled by a new high-fidelity pattern-searching and glycopeptide validation algorithm termed IsoStamp v2.0, as well as by novel stable isotope probes. Application of the IsoTaG platform to 15 cell lines metabolically labeled with Ac4GalNAz or Ac4ManNAz revealed 1375 N- and 2159 O-glycopeptides, variously modified with 74 discrete glycan structures. Glycopeptide-bound glycans observed by I...