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Maturing Glycoproteomics Technologies Provide Unique Structural Insights into the N-glycoproteome and Its Regulation in Health and Disease*

  • Morten Thaysen-Andersen
    Correspondence
    To whom correspondence should be addressed:Biomolecular Frontiers Research Centre, Department of Chemistry and Biomolecular Sciences, Macquarie University - Sydney, NSW 2109 Australia. Tel.:+61 2 9850 7487; Fax:+61 2 9850 6192;
    Affiliations
    Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, NSW, Australia;
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  • Nicolle H. Packer
    Affiliations
    Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, NSW, Australia;
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  • Benjamin L. Schulz
    Affiliations
    School of Chemistry & Molecular Biosciences, St Lucia, The University of Queensland, Brisbane, QLD, Australia
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  • Author Footnotes
    * M.T.-A. was supported by an Early Career Fellowship from the Cancer Institute, NSW, Australia and by a Macquarie University Research Development Grant (MQRDG). B.L.S. was supported by a National Health and Medical Research Council R.D. Wright Biomedical Career Development Fellowship Level 2 (APP1087975). N.P. acknowledges the funding of the Australian Research Council Centre of Excellence in Nanoscale Biophotonics (CE140100003).
    1 The abbreviations used are:OSToligosaccharyltransferaseDDAdata-dependent acquisitionDIAdata-independent acquisitionECDelectron capture dissociationERendoplasmic reticulumETDelectron transfer dissociationEThcDelectron transfer and higher-energy collision dissociationFDRfalse discovery rateFucfucoseGalNAcN-acetylgalactosamineGlcNAcN-acetylglucosamineHCDhigher-energy collision dissociationHILIChydrophilic interaction liquid chromatographyIRinsulin resistanceiTRAQisobaric tag for relative and absolute quantitationManmannoseMRMmultiple reaction monitoringNeuAcN-acetylneuraminic acidNeuGcN-glycolylneuraminic acidPGCporous graphitized carbonRPreversed phaseSILACstable isotope labelling with amino acids in cell cultureSPEsolid phase extractionXICextracted ion chromatogram.
Open AccessPublished:February 29, 2016DOI:https://doi.org/10.1074/mcp.O115.057638
      The glycoproteome remains severely understudied because of significant analytical challenges associated with glycoproteomics, the system-wide analysis of intact glycopeptides. This review introduces important structural aspects of protein N-glycosylation and summarizes the latest technological developments and applications in LC-MS/MS-based qualitative and quantitative N-glycoproteomics. These maturing technologies provide unique structural insights into the N-glycoproteome and its synthesis and regulation by complementing existing methods in glycoscience. Modern glycoproteomics is now sufficiently mature to initiate efforts to capture the molecular complexity displayed by the N-glycoproteome, opening exciting opportunities to increase our understanding of the functional roles of protein N-glycosylation in human health and disease.
      Protein glycosylation encompasses a broad class of post-translational modifications involving the covalent attachment of complex carbohydrates (glycans) to specific amino acid residues of polypeptide chains. The human biosynthetic machinery catalyzes diverse types of glycosylation, with the best studied being attachment of glycans to asparagine (N-glycosylation) and serine/threonine (O-glycosylation) residues (
      • Varki A.
      • Sharon N.
      Historical Background and Overview.
      ). As with all types of protein glycosylation, N-glycosylation is a template-less modification synthesized by a suite of glycosylation enzymes in the secretory pathway, Fig. 1A (
      • Shrimal S.
      • Cherepanova N.A.
      • Gilmore R.
      Cotranslational and posttranslocational N-glycosylation of proteins in the endoplasmic reticulum.
      ). Template-less synthesis means that glycosylation is determined by the physiological state of the glycosylation machinery and the nature of the proteins undergoing glycosylation. Jointly, these attributes determine the repertoire of glycans present on synthesized glycoproteins (glycoforms) and create the important features of protein site- and cell-specific glycosylation (
      • Thaysen-Andersen M.
      • Packer N.H.
      Site-specific glycoproteomics confirms that protein structure dictates formation of N-glycan type, core fucosylation and branching.
      ,
      • Rudd P.M.
      • Dwek R.A.
      Glycosylation: heterogeneity and the 3D structure of proteins.
      ,
      • Lee L.Y.
      • Lin C.H.
      • Fanayan S.
      • Packer N.H.
      • Thaysen-Andersen M.
      Differential site accessibility mechanistically explains subcellular-specific N-glycosylation determinants.
      ). Protein glycosylation is therefore a spatiotemporal dynamic modification that cells can utilize to respond to the constantly changing milieu.
      Figure thumbnail gr1
      Fig. 1.Overview of the biosynthesis and structural classes of mammalian protein N-glycosylation. A, Schematic summary of the biosynthetic machinery of N-glycoproteins. The enzymatic processing, which is initiated while the glycoproteins are still being translated, translocated, and folded, may terminate at any point in the enzymatic sequence depending partially on the Asn solvent accessibility of the maturely folded glycoprotein (
      • Thaysen-Andersen M.
      • Packer N.H.
      Site-specific glycoproteomics confirms that protein structure dictates formation of N-glycan type, core fucosylation and branching.
      ). This generates site-, cell-, and even subcellular-specific glycoform heterogeneity forming one of the functionally most important features of the glycoproteome (
      • Lee L.Y.
      • Lin C.H.
      • Fanayan S.
      • Packer N.H.
      • Thaysen-Andersen M.
      Differential site accessibility mechanistically explains subcellular-specific N-glycosylation determinants.
      ), and also creates substantial analytical challenges. TGN: trans-Golgi network. B, Mammalian N-glycoproteins are typically divided into three main N-glycan classes: high mannose, hybrid, and complex type. Unusual paucimannosidic and chitobiose core type N-glycans arising from unconventional truncation pathways (dashed box) have been reported in specific cell types and physiological conditions (
      • Thaysen-Andersen M.
      • Venkatakrishnan V.
      • Loke I.
      • Laurini C.
      • Diestel S.
      • Parker B.L.
      • Packer N.H.
      Human neutrophils secrete bioactive paucimannosidic proteins from azurophilic granules into pathogen-infected sputum.
      ). Monosaccharide residues are depicted according to the establish nomenclature (
      • Varki A.
      • Cummings R.D.
      • Aebi M.
      • Packer N.H.
      • Seeberger P.H.
      • Esko J.D.
      • Stanley P.
      • Hart G.
      • Darvill A.
      • Kinoshita T.
      • Prestegard J.J.
      • Schnaar R.L.
      • Freeze H.H.
      • Marth J.D.
      • Bertozzi C.R.
      • Etzler M.E.
      • Frank M.
      • Vliegenthart J.F.
      • Lütteke T.
      • Perez S.
      • Bolton E.
      • Rudd P.
      • Paulson J.
      • Kanehisa M.
      • Toukach P.
      • Aoki-Kinoshita K.F.
      • Dell A.
      • Narimatsu H.
      • York W.
      • Taniguchi N.
      • Kornfeld S.
      Symbol Nomenclature for Graphical Representations of Glycans.
      ) with residual monoisotopic masses provided.
      N-linked glycans are typically present on asparagine residues in AsnXxxSer/Thr, Xxx ≠ Pro consensus sequences (sequons) in humans. This preference is caused by specific recognition of the sequon by the peptide-binding site of an oligosaccharyltransferase (OST)
      The abbreviations used are:
      OST
      oligosaccharyltransferase
      DDA
      data-dependent acquisition
      DIA
      data-independent acquisition
      ECD
      electron capture dissociation
      ER
      endoplasmic reticulum
      ETD
      electron transfer dissociation
      EThcD
      electron transfer and higher-energy collision dissociation
      FDR
      false discovery rate
      Fuc
      fucose
      GalNAc
      N-acetylgalactosamine
      GlcNAc
      N-acetylglucosamine
      HCD
      higher-energy collision dissociation
      HILIC
      hydrophilic interaction liquid chromatography
      IR
      insulin resistance
      iTRAQ
      isobaric tag for relative and absolute quantitation
      Man
      mannose
      MRM
      multiple reaction monitoring
      NeuAc
      N-acetylneuraminic acid
      NeuGc
      N-glycolylneuraminic acid
      PGC
      porous graphitized carbon
      RP
      reversed phase
      SILAC
      stable isotope labelling with amino acids in cell culture
      SPE
      solid phase extraction
      XIC
      extracted ion chromatogram.
      (
      • Lizak C.
      • Gerber S.
      • Numao S.
      • Aebi M.
      • Locher K.P.
      X-ray structure of a bacterial oligosaccharyltransferase.
      ), the enzyme which catalyzes this reaction. However, it is now clear that mammalian cells also have the ability to rarely glycosylate more relaxed sequons (e.g. AsnXxxCys) (
      • Trinidad J.C.
      • Schoepfer R.
      • Burlingame A.L.
      • Medzihradszky K.F.
      N- and O-glycosylation in the murine synaptosome.
      ,
      • Zielinska D.F.
      • Gnad F.
      • Wiśniewski J.R.
      • Mann M.
      Precision mapping of an in vivo N-glycoproteome reveals rigid topological and sequence constraints.
      ,
      • Tan Z.
      • Yin H.
      • Nie S.
      • Lin Z.
      • Zhu J.
      • Ruffin M.T.
      • Anderson M.A.
      • Simeone D.M.
      • Lubman D.M.
      Large-scale identification of core-fucosylated glycopeptide sites in pancreatic cancer serum using mass spectrometry.
      ,
      • Asperger A.
      • Marx K.
      • Albers C.
      • Molin L.
      • Pinato O.
      Low abundant N-linked glycosylation in hen egg white lysozyme is localized at nonconsensus sites.
      ,
      • Faid V.
      • Denguir N.
      • Chapuis V.
      • Bihoreau N.
      • Chevreux G.
      Site-specific N-glycosylation analysis of human factor XI: Identification of a noncanonical NXC glycosite.
      ,
      • Chandler K.B.
      • Brnakova Z.
      • Sanda M.
      • Wang S.
      • Stalnaker S.H.
      • Bridger R.
      • Zhao P.
      • Wells L.
      • Edwards N.J.
      • Goldman R.
      Site-specific glycan microheterogeneity of inter-alpha-trypsin inhibitor heavy chain H4.
      ,
      • Medzihradszky K.F.
      • Kaasik K.
      • Chalkley R.J.
      Tissue-Specific Glycosylation at the Glycopeptide Level.
      ). The use of such nonconsensus sequons seems to be more frequent in rodents, where even glutamine-linked glycosylation has been reported (
      • Valliere-Douglass J.F.
      • Eakin C.M.
      • Wallace A.
      • Ketchem R.R.
      • Wang W.
      • Treuheit M.J.
      • Balland A.
      Glutamine-linked and non-consensus asparagine-linked oligosaccharides present in human recombinant antibodies define novel protein glycosylation motifs.
      ). Low efficiency N-glycosylation in these noncanonical sequons is consistent with a role of the canonical sequon in recognition and high-affinity binding to OST to promote glycan transfer (
      • Zacchi L.F.
      • Schulz B.L.
      N-glycoprotein macroheterogeneity: biological implications and proteomic characterization.
      ). Mammalian N-glycans share a common trimannosylchitobiose core comprised of three mannose (Man) and two N-acetylglucosamine (GlcNAc) residues, extended with a variety of monosaccharides including Man, GlcNAc, galactose (Gal), fucose (Fuc), N-acetylgalactosamine (GalNAc) and sialic acids such as N-acetylneuraminic acid (NeuAc) and N-glycolylneuraminic acid (NeuGc). N-glycans can be further modified by noncarbohydrate moieties including phosphorylation, sulfation and acetylation (
      • Moremen K.W.
      • Tiemeyer M.
      • Nairn A.V.
      Vertebrate protein glycosylation: diversity, synthesis and function.
      ,
      • Cummings R.D.
      The repertoire of glycan determinants in the human glycome.
      ). The conserved trimannosylchitobiose core of N-glycans is a remnant of the N-glycan precursor (Glc3Man9GlcNAc2) initially transferred to proteins in mammalian cells. This oligosaccharide structure is built stepwise on a dolichol pyrophosphate carrier embedded in the endoplasmic reticulum (ER) membrane, and is transferred en bloc to nascent polypeptides by OST. The terminal Glc and Man play critical roles in assisting glycoprotein folding and in ensuring glycoprotein quality control in the ER. After glycoproteins are correctly folded, the terminal Glc and Man are generally removed by α-glucosidases and α-mannosidases in the ER and cis-Golgi. N-glycans can then be extended by glycosyltransferases in the multiple Golgi compartments, potentially resulting in an extreme diversity of structures on mature glycoproteins in an organism-, cell-, or regulation-specific manner. The diverse mammalian N-glycans can be crudely classified into three conventional classes: high mannose, hybrid, and complex type, Fig. 1B. However, it is becoming clear that other unconventional N-glycan classes such as paucimannosidic and chitobiose core types decorate some mammalian glycoproteins (see below) (
      • Trinidad J.C.
      • Schoepfer R.
      • Burlingame A.L.
      • Medzihradszky K.F.
      N- and O-glycosylation in the murine synaptosome.
      ,
      • Medzihradszky K.F.
      • Kaasik K.
      • Chalkley R.J.
      Tissue-Specific Glycosylation at the Glycopeptide Level.
      ,
      • Venkatakrishnan V.
      • Thaysen-Andersen M.
      • Chen S.C.
      • Nevalainen H.
      • Packer N.H.
      Cystic fibrosis and bacterial colonization define the sputum N-glycosylation phenotype.
      ,
      • Dahmen A.C.
      • Fergen M.T.
      • Laurini C.
      • Schmitz B.
      • Loke I.
      • Thaysen-Andersen M.
      • Diestel S.
      Paucimannosidic glycoepitopes are functionally involved in proliferation of neural progenitor cells in the subventricular zone.
      ,
      • Loke I.
      • Packer N.H.
      • Thaysen-Andersen M.
      Complementary LC-MS/MS-based N-glycan, N-glycopeptide, and intact N-glycoprotein profiling reveals unconventional Asn71-glycosylation of human neutrophil cathepsin G.
      ,
      • Thaysen-Andersen M.
      • Venkatakrishnan V.
      • Loke I.
      • Laurini C.
      • Diestel S.
      • Parker B.L.
      • Packer N.H.
      Human neutrophils secrete bioactive paucimannosidic proteins from azurophilic granules into pathogen-infected sputum.
      ). The structures, biosynthetic pathways, and associated disorders of N-glycosylation have been recently reviewed elsewhere and readers are encouraged to use these resources for a deeper introduction (
      • Moremen K.W.
      • Tiemeyer M.
      • Nairn A.V.
      Vertebrate protein glycosylation: diversity, synthesis and function.
      ,
      • Freeze H.H.
      Understanding human glycosylation disorders: biochemistry leads the charge.
      ).
      A substantial proportion of mammalian genomes is dedicated to genes encoding proteins involved in glycosylation pathways, and these are highly conserved. Consistent with this, glycosylation is central to many biological processes. N-glycans are critical for enabling efficient glycoprotein folding and for maintaining the structural and functional integrity of glycoproteins (
      • Moremen K.W.
      • Tiemeyer M.
      • Nairn A.V.
      Vertebrate protein glycosylation: diversity, synthesis and function.
      ). Protein N-glycosylation is also intimately associated with development processes (
      • Haltiwanger R.S.
      • Lowe J.B.
      Role of glycosylation in development.
      ,
      • Tran D.T.
      • Ten Hagen K.G.
      Mucin-type O-glycosylation during development.
      ), with facilitating or preventing bacterial binding to the host (
      • Venkatakrishnan V.
      • Packer N.H.
      • Thaysen-Andersen M.
      Host mucin glycosylation plays a role in bacterial adhesion in lungs of individuals with cystic fibrosis.
      ) and with sustaining the normal function of individual cells, tissues, organs, and organisms (
      • Varki A.
      Biological roles of oligosaccharides: all of the theories are correct.
      ). Finally, protein glycosylation is a strictly regulated modification process in healthy cells, and these biochemical processes are dysregulated in various pathologies including, but not restricted to, cancer (
      • Christiansen M.N.
      • Chik J.
      • Lee L.
      • Anugraham M.
      • Abrahams J.L.
      • Packer N.H.
      Cell surface protein glycosylation in cancer.
      ,
      • Stuchlová Horynová M.
      • Raška M.
      • Clausen H.
      • Novak J.
      Aberrant O-glycosylation and anti-glycan antibodies in an autoimmune disease IgA nephropathy and breast adenocarcinoma.
      ), inflammation (
      • Scott D.W.
      • Patel R.P.
      Endothelial heterogeneity and adhesion molecules N-glycosylation: implications in leukocyte trafficking in inflammation.
      ), Alzheimer's disease (
      • Schedin-Weiss S.
      • Winblad B.
      • Tjernberg L.O.
      The role of protein glycosylation in Alzheimer disease.
      ), multiple sclerosis (
      • Grigorian A.
      • Mkhikian H.
      • Li C.F.
      • Newton B.L.
      • Zhou R.W.
      • Demetriou M.
      Pathogenesis of multiple sclerosis via environmental and genetic dysregulation of N-glycosylation.
      ), and cystic fibrosis (
      • Venkatakrishnan V.
      • Packer N.H.
      • Thaysen-Andersen M.
      Host mucin glycosylation plays a role in bacterial adhesion in lungs of individuals with cystic fibrosis.
      ). Disease-associated changes in protein glycosylation may arise from changes in glycoprotein abundance, glycosylation site occupancy (macro-heterogeneity or “glycosylation efficiency”), or glycan micro-heterogeneity at different sites on a glycoprotein (see details below). Changes in the glycoprotein micro-heterogeneity are dictated by the capacity of glycan-processing enzymes (glycosidases and glycosyltransferases) in the glycoprotein biosynthesis pathway, the nature of specific glycoprotein substrates, and other cellular factors (
      • Thaysen-Andersen M.
      • Packer N.H.
      Site-specific glycoproteomics confirms that protein structure dictates formation of N-glycan type, core fucosylation and branching.
      ,
      • Rudd P.M.
      • Dwek R.A.
      Glycosylation: heterogeneity and the 3D structure of proteins.
      ).
      This review summarizes the present analytical tools and technologies capable of performing large-scale (system-wide) analysis of protein N-glycosylation micro-heterogeneity and the unique structural insights that can be derived from such experiments by covering the very latest literature describing recent technological developments and applications in LC-MS/MS-based qualitative and quantitative N-glycoproteomics.

      System-wide Structural Analysis of Protein N-glycosylation

      Deciphering the glycosylation ‘code’ has been the ambition of generations of glycobiologists. The ability to accurately characterize the structure of glycoproteins is necessary if we are to succeed in our quest to unravel the diverse functions of glycans and develop the next generations of glycoprotein-based therapeutics (
      • Dalziel M.
      • Crispin M.
      • Scanlan C.N.
      • Zitzmann N.
      • Dwek R.A.
      Emerging principles for the therapeutic exploitation of glycosylation.
      ). However, glycoproteins are challenging to characterize because of the multiple layers of structural diversity that form a spectrum of chemically similar glycofoms. The information needed to unambiguously characterize such heterogeneous glycoproteins is therefore consequently much larger than for unmodified proteins or for proteins with structurally simple modifications such as phosphorylation or methylation. Even the most detailed modern glycoprotein characterization studies usually only capture part of the glycoprotein structure. Some glycoprotein structural features can be inferred or predicted from the biosynthetic constraints of the well-studied glycosylation machinery of mammalian cells (
      • Aebi M.
      N-linked protein glycosylation in the ER.
      ). Nevertheless, it is important to stress that even incomplete structural information can often be very useful in deciphering the structure/function relationships of glycoproteins, and in identifying alterations in the biosynthetic glycosylation machinery.
      Glycoproteomics is the site-specific analysis of the glycoproteome at the systems level, Fig. 2A. Glycoproteomics experimental workflows are typically initiated with protein extraction from biological samples, denaturation, and protease digestion, Fig. 2B. At this step, isotopic labels assisting in glycopeptide quantitation or enhancing their MS features (e.g. ionization and fragmentation) can be introduced. The resulting peptide mixtures are often extremely complex and glycopeptides are consequently typically enriched and/or prefractionated prior to detection, usually by LC-MS/MS. Glycoproteomics experiments are commonly based on the identification, and less frequently, also the quantitation of intact glycopeptides. Glycoproteomics yields system-wide information on the glycoprotein carriers, the glycan attachment sites, the occupancies of glycosylation at each site, and the structure and heterogeneity of the attached glycans. As showcased in this review by recent examples, glycoproteomics is a powerful technology to map disease-associated alterations in the glycoproteome, Fig. 3. Such glycosylation alterations may originate from multiple tissues, which may be differently regulated during pathogenesis. Typically, in glycoproteomics investigations, intact glycopeptides derived from the total complement of glycoproteins extracted from bodily fluids or complex tissues from healthy and diseased individuals (or other biological scenarios) are qualitatively and quantitatively compared. By also measuring any changes in glycoprotein abundance and site occupancy, the exact mechanism(s) contributing to the observed glycoproteome regulation can be interrogated (see example below).
      Figure thumbnail gr2
      Fig. 2.A, Definitions and explanations of commonly used nomenclature in glycoproteomics. B, Generic workflow illustrating important components of a glycoproteomics experiment, which crudely can be divided into segments related to glycopeptide sample preparation (top box) and detection (bottom box). Typical examples of the individual components are provided. *Additional sample handling including glycoprotein derivatization and glycopeptide labeling for quantitation purposes may be introduced at this step.
      Figure thumbnail gr3
      Fig. 3.Three fundamental levels of molecular dysregulation of multiple tissues contributing to an altered secreted N-glycoproteome during disease. A, Hypothetical example illustrating three sources of dysregulation: 1) protein level (green), 2) site occupancy (blue), or 3) glycosylation micro-heterogeneity (red) from three separate tissues (Tissue A-C) contributing to a joint secreted N-glycoproteome (Protein A-C) in a body fluid derived from disease (right) and ‘normal’ healthy (left) condition. B, After proteolysis and enrichment, the altered abundance of the resulting glycopeptides can be detected using LC-MS/MS-based label-free quantitative glycoproteomics as shown by color-coded traces representing extracted ion chromatograms (XICs). However, establishing which of the three mechanisms causes the glycopeptide alterations for the detected glycoproteins may be challenging solely with glycopeptide analysis, especially in glycoproteomes arising from multiple tissues. Parallel quantitative proteomics and “deglycoproteomics” (detection of formerly occupied N-sites) of the same samples can assist in this task.
      Many other analytical approaches can be used to characterize aspects of glycoprotein structural diversity. These include site-specific analysis of N-glycoproteins isolated to relative purity rather than in complex mixtures (
      • Faid V.
      • Denguir N.
      • Chapuis V.
      • Bihoreau N.
      • Chevreux G.
      Site-specific N-glycosylation analysis of human factor XI: Identification of a noncanonical NXC glycosite.
      ,
      • Chandler K.B.
      • Brnakova Z.
      • Sanda M.
      • Wang S.
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      • Bridger R.
      • Zhao P.
      • Wells L.
      • Edwards N.J.
      • Goldman R.
      Site-specific glycan microheterogeneity of inter-alpha-trypsin inhibitor heavy chain H4.
      ,
      • Loke I.
      • Packer N.H.
      • Thaysen-Andersen M.
      Complementary LC-MS/MS-based N-glycan, N-glycopeptide, and intact N-glycoprotein profiling reveals unconventional Asn71-glycosylation of human neutrophil cathepsin G.
      ,
      • Song E.
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      Characterization of the glycosylation site of human PSA prompted by missense mutation using LC-MS/MS.
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      Direct site-specific glycoform identification and quantitative comparison of glycoprotein therapeutics: imiglucerase and velaglucerase alfa.
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      N-glycosylation characterization by liquid chromatography with mass spectrometry.
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      Recombinant human heterodimeric IL-15 complex displays extensive and reproducible N- and O-linked glycosylation.
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      Site-specific protein N- and O-glycosylation analysis by a C18-porous graphitized carbon-liquid chromatography-electrospray ionization mass spectrometry approach using pronase treated glycopeptides.
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      A microarray-matrix-assisted laser desorption/ionization-mass spectrometry approach for site-specific protein N-glycosylation analysis, as demonstrated for human serum immunoglobulin M (IgM).
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      Glycoproteomic studies of IgE from a novel hyper IgE syndrome linked to PGM3 mutation.
      ), N-glycomics analyses of glycans released from glycoproteins (
      • Venkatakrishnan V.
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      • Chen S.C.
      • Nevalainen H.
      • Packer N.H.
      Cystic fibrosis and bacterial colonization define the sputum N-glycosylation phenotype.
      ,
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      • Roser J.
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      Recombinant human heterodimeric IL-15 complex displays extensive and reproducible N- and O-linked glycosylation.
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      Automating mass spectrometry-based quantitative glycomics using aminoxy tandem mass tag reagents with SimGlycan.
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      In-depth N-glycome profiling of paired colorectal cancer and non-tumorigenic tissues reveals cancer-, stage- and EGFR-specific protein N-glycosylation.
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      Clinical glycomics employing graphitized carbon liquid chromatography-mass spectrometry.
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      Integrated proteomic and glycoproteomic analyses of prostate cancer cells reveals glycoprotein alteration in protein abundance and glycosylation.
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      • Netter H.J.
      Modification of asparagine-linked glycan density for the design of Hepatitis B virus virus-like particles with enhanced immunogenicity.
      ), and identification and quantification of previously glycosylated sites on de-N-glycosylated proteins (“deglycoproteomics”) after removal of the entire glycan or with remnant N-glycan core remaining (
      • Huang J.
      • Qin H.
      • Sun Z.
      • Huang G.
      • Mao J.
      • Cheng K.
      • Zhang Z.
      • Wan H.
      • Yao Y.
      • Dong J.
      • Zhu J.
      • Wang F.
      • Ye M.
      • Zou H.
      A peptide N-terminal protection strategy for comprehensive glycoproteome analysis using hydrazide chemistry based method.
      ,
      • DeCoux A.
      • Tian Y.
      • DeLeon-Pennell K.Y.
      • Nguyen N.T.
      • de Castro Brás L.E.
      • Flynn E.R.
      • Cannon P.L.
      • Griswold M.E.
      • Jin Y.F.
      • Puskarich M.A.
      • Jones A.E.
      • Lindsey M.L.
      Plasma glycoproteomics reveals sepsis outcomes linked to distinct proteins in common pathways.
      ,
      • Sugahara D.
      • Tomioka A.
      • Sato T.
      • Narimatsu H.
      • Kaji H.
      Large-scale identification of secretome glycoproteins recognized by Wisteria floribunda agglutinin: A glycoproteomic approach to biomarker discovery.
      ,
      • Pan S.
      Quantitative glycoproteomics for N-glycoproteome profiling.
      ,
      • Hill J.J.
      • Tremblay T.L.
      • Fauteux F.
      • Li J.
      • Wang E.
      • Aguilar-Mahecha A.
      • Basik M.
      • O'Connor-McCourt M.
      Glycoproteomic comparison of clinical triple-negative and luminal breast tumors.
      ,
      • Sok Hwee Cheow E.
      • Hwan Sim K.
      • de Kleijn D.
      • Neng Lee C.
      • Sorokin V.
      • Sze S.K.
      Simultaneous enrichment of plasma soluble and extracellular vesicular glycoproteins using prolonged ultracentrifugation-electrostatic repulsion-hydrophilic interaction chromatography (PUC-ERLIC) approach.
      ,
      • Feng M.
      • Fang Y.
      • Han B.
      • Xu X.
      • Fan P.
      • Hao Y.
      • Qi Y.
      • Hu H.
      • Huo X.
      • Meng L.
      • Wu B.
      • Li J.
      In-depth N-glycosylation reveals species-specific modifications and functions of the royal jelly protein from Western (Apis mellifera) and Eastern Honeybees (Apis cerana).
      ,
      • DeLeon-Pennell K.Y.
      • Tian Y.
      • Zhang B.
      • Cates C.A.
      • Padmanabhan Iyer R.
      • Cannon P.
      • Shah P.
      • Aiyetan P.
      • Halade G.V.
      • Ma Y.
      • Flynn E.
      • Zhang Z.
      • Jin Y.F.
      • Zhang H.
      • Lindsey M.L.
      CD36 is a matrix metalloproteinase-9 substrate that stimulates neutrophil apoptosis and removal during cardiac remodeling.
      ,
      • Huang J.
      • Wan H.
      • Yao Y.
      • Li J.
      • Cheng K.
      • Mao J.
      • Chen J.
      • Wang Y.
      • Qin H.
      • Zhang W.
      • Ye M.
      • Zou H.
      Highly efficient release of glycopeptides from hydrazide beads by hydroxylamine assisted PNGase F deglycosylation for N-glycoproteome analysis.
      ,
      • Clark D.J.
      • Mei Y.
      • Sun S.
      • Zhang H.
      • Yang A.J.
      • Mao L.
      Glycoproteomic approach identifies KRAS as a positive regulator of CREG1 in non-small cell lung cancer cells.
      ). Although these studies per se do not qualify under our definition of glycoproteomics, (site-specific analysis of the glycoproteome at the intact glycopeptide level), they still provide useful information in conjunction with glycoproteomics for the glycobiologist, provided correct experimental design is applied (
      • Palmisano G.
      • Melo-Braga M.N.
      • Engholm-Keller K.
      • Parker B.L.
      • Larsen M.R.
      Chemical deamidation: a common pitfall in large-scale N-linked glycoproteomic mass spectrometry-based analyses.
      ).

      Key Technologies and Recent Analytical Developments in N-glycoproteomics

      In 2014, we reviewed the status of modern LC-MS/MS-based glycoproteomics (
      • Thaysen-Andersen M.
      • Packer N.H.
      Advances in LC-MS/MS-based glycoproteomics: getting closer to system-wide site-specific mapping of the N- and O-glycoproteome.
      ). Other excellent recent reviews are also available (
      • Zacchi L.F.
      • Schulz B.L.
      N-glycoprotein macroheterogeneity: biological implications and proteomic characterization.
      ,
      • Schedin-Weiss S.
      • Winblad B.
      • Tjernberg L.O.
      The role of protein glycosylation in Alzheimer disease.
      ,
      • Pagel O.
      • Loroch S.
      • Sickmann A.
      • Zahedi R.P.
      Current strategies and findings in clinically relevant post-translational modification-specific proteomics.
      ,
      • Kolli V.
      • Schumacher K.N.
      • Dodds E.D.
      Engaging challenges in glycoproteomics: recent advances in MS-based glycopeptide analysis.
      ,
      • Baycin Hizal D.
      • Wolozny D.
      • Colao J.
      • Jacobson E.
      • Tian Y.
      • Krag S.S.
      • Betenbaugh M.J.
      • Zhang H.
      Glycoproteomic and glycomic databases.
      ,
      • Zhu Z.
      • Desaire H.
      Carbohydrates on proteins: site-specific glycosylation analysis by mass spectrometry.
      ,
      • Lazar I.M.
      • Deng J.
      • Ikenishi F.
      • Lazar A.C.
      Exploring the glycoproteomics landscape with advanced MS technologies.
      ,
      • Thaysen-Andersen M.
      • Larsen M.R.
      • Packer N.H.
      • Palmisano G.
      Structural analysis of glycoprotein sialylation - Part I: pre-LC-MS analytical strategies.
      ,
      • Palmisano G.
      • Larsen M.R.
      • Packer N.H.
      • Thaysen-Andersen M.
      Structural analysis of glycoprotein sialylation -part II: LC-MS based detection.
      ,
      • Moh E.S.
      • Thaysen-Andersen M.
      • Packer N.H.
      Relative versus absolute quantitation in disease glycomics.
      ,
      • Miura Y.
      • Endo T.
      Glycomics and glycoproteomics focused on aging and age-related diseases - Glycans as a potential biomarker for physiological alterations.
      ). Thus, this review will highlight the very latest (∼2014–present) technological advances and applications in glycoproteomics, which have been instrumental for the recent performance improvements in detection limits, accuracy of glycopeptide identification and quantitation, and gains in glycoproteome coverage.
      The modern discipline of glycoproteomics has deep roots in the protein and carbohydrate analytical chemistry pioneered in the late 1980s and early 1990s with the advent of biomolecular mass spectrometry (
      • Burlingame A.L.
      Characterization of protein glycosylation by mass spectrometry.
      ). Impressive analytical strategies using relatively insensitive MS instrumentation were developed e.g. the selective detection of glycopeptides in mixtures using deglycosylation-based mass shifts (
      • Carr S.A.
      • Roberts G.D.
      Carbohydrate mapping by mass spectrometry: a novel method for identifying attachment sites of Asn-linked sugars in glycoproteins.
      ) and selection ion chromatograms (
      • Huddleston M.J.
      • Bean M.F.
      • Carr S.A.
      Collisional fragmentation of glycopeptides by electrospray ionization LC/MS and LC/MS/MS: methods for selective detection of glycopeptides in protein digests.
      ), and the fundamentals of glycopeptide ionization and fragmentation behavior were accurately described (
      • Küster B.
      • Naven T.J.
      • Harvey D.J.
      Effect of the reducing-terminal substituents on the high energy collision-induced dissociation matrix-assisted laser desorption/ionization mass spectra of oligosaccharides.
      ,
      • Townsend R.R.
      • Heller D.N.
      • Fenselau C.C.
      • Lee Y.C.
      Determination of the sialylation pattern of human fibrinogen glycopeptides with fast atom bombardment.
      ). These early studies remain a solid foundation on which many modern glycoanalytical strategies are conceived. It is also clear that glycoproteomics more recently has profited handsomely from technology developments arising from the larger and more mature discipline of proteomics including sample handling, LC-MS/MS acquisition strategies, and data handling and processing (
      • Richards A.L.
      • Merrill A.E.
      • Coon J.J.
      Proteome sequencing goes deep.
      ). In parallel, glycoproteomics has been a beneficiary of the continual performance enhancements of modern mass spectrometers including improved speed, sensitivity, resolution, and accuracy, most notably implemented on the latest Q-TOF (Sciex, Waters, Agilent, Bruker) and on multiple Orbitrap (Thermo) instrument platforms (
      • Peng W.P.
      • Chou S.W.
      • Patil A.A.
      Measuring masses of large biomolecules and bioparticles using mass spectrometric techniques.
      ). Developments and applications of several key glycoproteomics-specific technologies have additionally been critical for the rapid maturation of glycoproteomics workflows over the past two years, Table I. Specifically, key advances have been made in the enrichment of intact glycopeptides from complex peptide mixtures, in LC-MS/MS-based detection of intact glycopeptides through optimized dissociation and acquisition styles of glycopeptides, and in data handling for more automated, yet still confident, glycopeptide identification and quantitation.
      Table ILatest developments and applications of the key components in N-glycoproteomics
      Component of glycoproteomics workflowReferences
      Sample preparation of N-glycopeptides
      EnrichmentHILIC SPE(
      • Thaysen-Andersen M.
      • Venkatakrishnan V.
      • Loke I.
      • Laurini C.
      • Diestel S.
      • Parker B.L.
      • Packer N.H.
      Human neutrophils secrete bioactive paucimannosidic proteins from azurophilic granules into pathogen-infected sputum.
      ,
      • Liu J.
      • Wang F.
      • Zhu J.
      • Mao J.
      • Liu Z.
      • Cheng K.
      • Qin H.
      • Zou H.
      Highly efficient N-glycoproteomic sample preparation by combining C(18) and graphitized carbon adsorbents.
      ,
      • Chen R.
      • Seebun D.
      • Ye M.
      • Zou H.
      • Figeys D.
      Site-specific characterization of cell membrane N-glycosylation with integrated hydrophilic interaction chromatography solid phase extraction and LC-MS/MS.
      ,
      • Dedvisitsakul P.
      • Jacobsen S.
      • Svensson B.
      • Bunkenborg J.
      • Finnie C.
      • Hägglund P.
      Glycopeptide enrichment using a combination of ZIC-HILIC and cotton wool for exploring the glycoproteome of wheat flour albumins.
      ,
      • Parker B.L.
      • Thaysen-Andersen M.
      • Fazakerley D.J.
      • Holliday M.
      • Packer N.H.
      • James D.E.
      Terminal galactosylation and sialylation switching on membrane glycoproteins upon TNF-alpha-induced insulin resistance in adipocytes.
      ,
      • Parker B.L.
      • Thaysen-Andersen M.
      • Solis N.
      • Scott N.E.
      • Larsen M.R.
      • Graham M.E.
      • Packer N.H.
      • Cordwell S.J.
      Site-specific glycan-Peptide analysis for determination of N-glycoproteome heterogeneity.
      ,
      • Noro E.
      • Togayachi A.
      • Sato T.
      • Tomioka A.
      • Fujita M.
      • Sukegawa M.
      • Suzuki N.
      • Kaji H.
      • Narimatsu H.
      Large-scale identification of N-glycan glycoproteins carrying lewis x and site-specific N-glycan alterations in Fut9 Knockout mice.
      ,
      • Cao L.
      • Tolić N.
      • Qu Y.
      • Meng D.
      • Zhao R.
      • Zhang Q.
      • Moore R.J.
      • Zink E.M.
      • Lipton M.S.
      • Paša-Tolić L.
      • Wu S.
      Characterization of intact N- and O-linked glycopeptides using higher energy collisional dissociation.
      ,
      • Zhao Y.
      • Szeto S.S.
      • Kong R.P.
      • Law C.H.
      • Li G.
      • Quan Q.
      • Zhang Z.
      • Wang Y.
      • Chu I.K.
      Online two-dimensional porous graphitic carbon/reversed phase liquid chromatography platform applied to shotgun proteomics and glycoproteomics.
      ,
      • Liu M.
      • Zhang Y.
      • Chen Y.
      • Yan G.
      • Shen C.
      • Cao J.
      • Zhou X.
      • Liu X.
      • Zhang L.
      • Shen H.
      • Lu H.
      • He F.
      • Yang P.
      Efficient and accurate glycopeptide identification pipeline for high-throughput site-specific N-glycosylation analysis.
      ,
      • Lynn K.S.
      • Chen C.C.
      • Lih T.M.
      • Cheng C.W.
      • Su W.C.
      • Chang C.H.
      • Cheng C.Y.
      • Hsu W.L.
      • Chen Y.J.
      • Sung T.Y.
      MAGIC: an automated N-linked glycoprotein identification tool using a Y1-ion pattern matching algorithm and in silico MS(2) approach.
      ,
      • Goyallon A.
      • Cholet S.
      • Chapelle M.
      • Junot C.
      • Fenaille F.
      Evaluation of a combined glycomics and glycoproteomics approach for studying the major glycoproteins present in biofluids: Application to cerebrospinal fluid.
      ,
      • Mayampurath A.
      • Song E.
      • Mathur A.
      • Yu C.Y.
      • Hammoud Z.
      • Mechref Y.
      • Tang H.
      Label-free glycopeptide quantification for biomarker discovery in human sera.
      ,
      • Ma C.
      • Zhang Q.
      • Qu J.
      • Zhao X.
      • Li X.
      • Liu Y.
      • Wang P.G.
      A precise approach in large scale core-fucosylated glycoprotein identification with low- and high-normalized collision energy.
      ,
      • Cao Q.
      • Zhao X.
      • Zhao Q.
      • Lv X.
      • Ma C.
      • Li X.
      • Zhao Y.
      • Peng B.
      • Ying W.
      • Qian X.
      Strategy integrating stepped fragmentation and glycan diagnostic ion-based spectrum refinement for the identification of core fucosylated glycoproteome using mass spectrometry.
      ,
      • Cheng K.
      • Chen R.
      • Seebun D.
      • Ye M.
      • Figeys D.
      • Zou H.
      Large-scale characterization of intact N-glycopeptides using an automated glycoproteomic method.
      ,
      • Scott N.E.
      • Marzook N.B.
      • Cain J.A.
      • Solis N.
      • Thaysen-Andersen M.
      • Djordjevic S.P.
      • Packer N.H.
      • Larsen M.R.
      • Cordwell S.J.
      Comparative proteomics and glycoproteomics reveal increased N-linked glycosylation and relaxed sequon specificity in Campylobacter jejuni NCTC11168 O.
      )
      Synthetic polymer mixed-mode HILIC SPE(
      • Pan Y.
      • Ma C.
      • Tong W.
      • Fan C.
      • Zhang Q.
      • Zhang W.
      • Tian F.
      • Peng B.
      • Qin W.
      • Qian X.
      Preparation of sequence-controlled triblock copolymer-grafted silica microparticles by sequential-ATRP for highly efficient glycopeptides enrichment.
      ,
      • Huang G.
      • Xiong Z.
      • Qin H.
      • Zhu J.
      • Sun Z.
      • Zhang Y.
      • Peng X.
      • ou J.
      • Zou H.
      Synthesis of zwitterionic polymer brushes hybrid silica nanoparticles via controlled polymerization for highly efficient enrichment of glycopeptides.
      ,
      • Bodnar E.D.
      • Perreault H.
      Synthesis and evaluation of carboxymethyl chitosan for glycopeptide enrichment.
      ,
      • Zheng J.
      • Xiao Y.
      • Wang L.
      • Lin Z.
      • Yang H.
      • Zhang L.
      • Chen G.
      Click synthesis of glucose-functionalized hydrophilic magnetic mesoporous nanoparticles for highly selective enrichment of glycopeptides and glycans.
      ,
      • Yang S.
      • Mishra S.
      • Chen L.
      • Zhou J.Y.
      • Chan D.W.
      • Chatterjee S.
      • Zhang H.
      Integrated glycoprotein immobilization method for glycopeptide and glycan analysis of cardiac hypertrophy.
      )
      Lectin SPE/chromatography(
      • Trinidad J.C.
      • Schoepfer R.
      • Burlingame A.L.
      • Medzihradszky K.F.
      N- and O-glycosylation in the murine synaptosome.
      ,
      • Tan Z.
      • Yin H.
      • Nie S.
      • Lin Z.
      • Zhu J.
      • Ruffin M.T.
      • Anderson M.A.
      • Simeone D.M.
      • Lubman D.M.
      Large-scale identification of core-fucosylated glycopeptide sites in pancreatic cancer serum using mass spectrometry.
      ,
      • Medzihradszky K.F.
      • Kaasik K.
      • Chalkley R.J.
      Tissue-Specific Glycosylation at the Glycopeptide Level.
      ,
      • Zhu F.
      • Trinidad J.C.
      • Clemmer D.E.
      Glycopeptide site heterogeneity and structural diversity determined by combined lectin affinity chromatography/IMS/CID/MS techniques.
      ,
      • Pagel O.
      • Loroch S.
      • Sickmann A.
      • Zahedi R.P.
      Current strategies and findings in clinically relevant post-translational modification-specific proteomics.
      ,
      • Saraswat M.
      • Joenvaara S.
      • Musante L.
      • Peltoniemi H.
      • Holthofer H.
      • Renkonen R.
      N-linked (N-) glycoproteomics of urimary exosomes.
      ,
      • Kontro H.
      • Joenväärä S.
      • Haglund C.
      • Renkonen R.
      Comparison of sialylated N-glycopeptide levels in serum of pancreatic cancer patients, acute pancreatitis patients, and healthy controls.
      ,
      • Noro E.
      • Togayachi A.
      • Sato T.
      • Tomioka A.
      • Fujita M.
      • Sukegawa M.
      • Suzuki N.
      • Kaji H.
      • Narimatsu H.
      Large-scale identification of N-glycan glycoproteins carrying lewis x and site-specific N-glycan alterations in Fut9 Knockout mice.
      ,
      • Zhao Y.
      • Szeto S.S.
      • Kong R.P.
      • Law C.H.
      • Li G.
      • Quan Q.
      • Zhang Z.
      • Wang Y.
      • Chu I.K.
      Online two-dimensional porous graphitic carbon/reversed phase liquid chromatography platform applied to shotgun proteomics and glycoproteomics.
      ,
      • Ruiz-May E.
      • Hucko S.
      • Howe K.J.
      • Zhang S.
      • Sherwood R.W.
      • Thannhauser T.W.
      • Rose J.K.
      A comparative study of lectin affinity based plant N-glycoproteome profiling using tomato fruit as a model.
      ,
      • Song E.
      • Zhu R.
      • Hammoud Z.T.
      • Mechref Y.
      LC-MS/MS quantitation of esophagus disease blood serum glycoproteins by enrichment with hydrazide chemistry and lectin affinity chromatography.
      ,
      • Mayampurath A.
      • Song E.
      • Mathur A.
      • Yu C.Y.
      • Hammoud Z.
      • Mechref Y.
      • Tang H.
      Label-free glycopeptide quantification for biomarker discovery in human sera.
      ,
      • Cao Q.
      • Zhao X.
      • Zhao Q.
      • Lv X.
      • Ma C.
      • Li X.
      • Zhao Y.
      • Peng B.
      • Ying W.
      • Qian X.
      Strategy integrating stepped fragmentation and glycan diagnostic ion-based spectrum refinement for the identification of core fucosylated glycoproteome using mass spectrometry.
      )
      Hydrazide chemistry
      Hydrazide is usually used to capture N-glycopeptides with a subsequent peptide N-glycosidase F release of formerly N-glycosylated peptides for glycosylation site mapping and is, thus, not a generally used tool in glycoproteomics.
      (
      • Huang J.
      • Qin H.
      • Sun Z.
      • Huang G.
      • Mao J.
      • Cheng K.
      • Zhang Z.
      • Wan H.
      • Yao Y.
      • Dong J.
      • Zhu J.
      • Wang F.
      • Ye M.
      • Zou H.
      A peptide N-terminal protection strategy for comprehensive glycoproteome analysis using hydrazide chemistry based method.
      ,
      • Hill J.J.
      • Tremblay T.L.
      • Fauteux F.
      • Li J.
      • Wang E.
      • Aguilar-Mahecha A.
      • Basik M.
      • O'Connor-McCourt M.
      Glycoproteomic comparison of clinical triple-negative and luminal breast tumors.
      ,
      • Song E.
      • Zhu R.
      • Hammoud Z.T.
      • Mechref Y.
      LC-MS/MS quantitation of esophagus disease blood serum glycoproteins by enrichment with hydrazide chemistry and lectin affinity chromatography.
      ,
      • Cao Q.
      • Ma C.
      • Bai H.
      • Li X.
      • Yan H.
      • Zhao Y.
      • Ying W.
      • Qian X.
      Multivalent hydrazide-functionalized magnetic nanoparticles for glycopeptide enrichment and identification.
      )
      Boronic acid magnetic beads(
      • Wang J.
      • Wang Y.
      • Gao M.
      • Zhang X.
      • Yang P.
      Multilayer hydrophilic poly(phenol-formaldehyde resin)-coated magnetic graphene for boronic acid immobilization as a novel matrix for glycoproteome analysis.
      ,
      • Wang M.
      • Zhang X.
      • Deng C.
      Facile synthesis of magnetic poly(styrene-co-4-vinylbenzene-boronic acid) microspheres for selective enrichment of glycopeptides.
      ,
      • Chen W.
      • Smeekens J.M.
      • Wu R.
      A universal chemical enrichment method for mapping the yeast N-glycoproteome by mass spectrometry (MS).
      )
      Acetone precipitation(
      • Takakura D.
      • Harazono A.
      • Hashii N.
      • Kawasaki N.
      Selective glycopeptide profiling by acetone enrichment and LC/MS.
      ,
      • Takakura D.
      • Tada M.
      • Kawasaki N.
      Membrane glycoproteomics of fetal lung fibroblasts using LC/MS.
      )
      Porous graphitized carbon (PGC)-RP SPE(
      • Liu J.
      • Wang F.
      • Zhu J.
      • Mao J.
      • Liu Z.
      • Cheng K.
      • Qin H.
      • Zou H.
      Highly efficient N-glycoproteomic sample preparation by combining C(18) and graphitized carbon adsorbents.
      ,
      • Ruiz-May E.
      • Hucko S.
      • Howe K.J.
      • Zhang S.
      • Sherwood R.W.
      • Thannhauser T.W.
      • Rose J.K.
      A comparative study of lectin affinity based plant N-glycoproteome profiling using tomato fruit as a model.
      )
      Size exclusion chromatography(
      • Saraswat M.
      • Joenväärä S.
      • Tomar A.K.
      • Singh S.
      • Yadav S.
      • Renkonen R.J.
      N-glycoproteomics of human seminal plasma glycoproteins.
      ,
      • Saraswat M.
      • Joenvaara S.
      • Musante L.
      • Peltoniemi H.
      • Holthofer H.
      • Renkonen R.
      N-linked (N-) glycoproteomics of urimary exosomes.
      )
      Metabolic labelling and enrichment(
      • Woo C.M.
      • Iavarone A.T.
      • Spiciarich D.R.
      • Palaniappan K.K.
      • Bertozzi C.R.
      Isotope-targeted glycoproteomics (IsoTaG): a mass-independent platform for intact N- and O-glycopeptide discovery and analysis.
      ,
      • Smeekens J.M.
      • Chen W.
      • Wu R.
      Mass spectrometric analysis of the cell surface N-glycoproteome by combining metabolic labeling and click chemistry.
      )
      Separation/fractionationNeutral/high pH RP-LC(
      • Trinidad J.C.
      • Schoepfer R.
      • Burlingame A.L.
      • Medzihradszky K.F.
      N- and O-glycosylation in the murine synaptosome.
      ,
      • Medzihradszky K.F.
      • Kaasik K.
      • Chalkley R.J.
      Tissue-Specific Glycosylation at the Glycopeptide Level.
      ,
      • Shah P.
      • Wang X.
      • Yang W.
      • Toghi Eshghi S.
      • Sun S.
      • Hoti N.
      • Chen L.
      • Yang S.
      • Pasay J.
      • Rubin A.
      • Zhang H.
      Integrated proteomic and glycoproteomic analyses of prostate cancer cells reveals glycoprotein alteration in protein abundance and glycosylation.
      ,
      • Parker B.L.
      • Thaysen-Andersen M.
      • Fazakerley D.J.
      • Holliday M.
      • Packer N.H.
      • James D.E.
      Terminal galactosylation and sialylation switching on membrane glycoproteins upon TNF-alpha-induced insulin resistance in adipocytes.
      ,
      • Parker B.L.
      • Thaysen-Andersen M.
      • Solis N.
      • Scott N.E.
      • Larsen M.R.
      • Graham M.E.
      • Packer N.H.
      • Cordwell S.J.
      Site-specific glycan-Peptide analysis for determination of N-glycoproteome heterogeneity.
      ,
      • Yang S.
      • Mishra S.
      • Chen L.
      • Zhou J.Y.
      • Chan D.W.
      • Chatterjee S.
      • Zhang H.
      Integrated glycoprotein immobilization method for glycopeptide and glycan analysis of cardiac hypertrophy.
      ,
      • Ruiz-May E.
      • Hucko S.
      • Howe K.J.
      • Zhang S.
      • Sherwood R.W.
      • Thannhauser T.W.
      • Rose J.K.
      A comparative study of lectin affinity based plant N-glycoproteome profiling using tomato fruit as a model.
      ,
      • Toghi Eshghi S.
      • Shah P.
      • Yang W.
      • Li X.
      • Zhang H.
      GPQuest: a spectral library matching algorithm for site-specific assignment of tandem mass spectra to intact N-glycopeptides.
      )
      2D (SCX/RP) -LC (online)(
      • Liu M.
      • Zhang Y.
      • Chen Y.
      • Yan G.
      • Shen C.
      • Cao J.
      • Zhou X.
      • Liu X.
      • Zhang L.
      • Shen H.
      • Lu H.
      • He F.
      • Yang P.
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      DDA-PD (m/z 204.086) ETD or ion trap CID/ETD(
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      Byonic(
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      • Haglund C.
      • Renkonen R.
      Comparison of sialylated N-glycopeptide levels in serum of pancreatic cancer patients, acute pancreatitis patients, and healthy controls.
      )
      Sweet-Heart(
      • Wu S.W.
      • Pu T.H.
      • Viner R.
      • Khoo K.H.
      Novel LC-MS(2) product dependent parallel data acquisition function and data analysis workflow for sequencing and identification of intact glycopeptides.
      ,
      • Wu S.W.
      • Liang S.Y.
      • Pu T.H.
      • Chang F.Y.
      • Khoo K.H.
      Sweet-Heart - an integrated suite of enabling computational tools for automated MS2/MS3 sequencing and identification of glycopeptides.
      ,
      • Liang S.Y.
      • Wu S.W.
      • Pu T.H.
      • Chang F.Y.
      • Khoo K.H.
      An adaptive workflow coupled with Random Forest algorithm to identify intact N-glycopeptides detected from mass spectrometry.
      )
      GlycoFragWork(
      • Mayampurath A.
      • Yu C.Y.
      • Song E.
      • Balan J.
      • Mechref Y.
      • Tang H.
      Computational framework for identification of intact glycopeptides in complex samples.
      ,
      • Mayampurath A.
      • Song E.
      • Mathur A.
      • Yu C.Y.
      • Hammoud Z.
      • Mechref Y.
      • Tang H.
      Label-free glycopeptide quantification for biomarker discovery in human sera.
      )
      GlycoMod(
      • Zhu F.
      • Trinidad J.C.
      • Clemmer D.E.
      Glycopeptide site heterogeneity and structural diversity determined by combined lectin affinity chromatography/IMS/CID/MS techniques.
      ,
      • Ruiz-May E.
      • Hucko S.
      • Howe K.J.
      • Zhang S.
      • Sherwood R.W.
      • Thannhauser T.W.
      • Rose J.K.
      A comparative study of lectin affinity based plant N-glycoproteome profiling using tomato fruit as a model.
      )
      Less used programs (i. e. GlycoMaster DB GRIP, MAGIC, GlycoPep Evaluator)(
      • Zhu Z.
      • Su X.
      • Go E.P.
      • Desaire H.
      New glycoproteomics software, GlycoPep Evaluator, generates decoy glycopeptides de novo and enables accurate false discovery rate analysis for small data sets.
      ,
      • Liu M.
      • Zhang Y.
      • Chen Y.
      • Yan G.
      • Shen C.
      • Cao J.
      • Zhou X.
      • Liu X.
      • Zhang L.
      • Shen H.
      • Lu H.
      • He F.
      • Yang P.
      Efficient and accurate glycopeptide identification pipeline for high-throughput site-specific N-glycosylation analysis.
      ,
      • Jansen B.C.
      • Reiding K.R.
      • Bondt A.
      • Hipgrave Ederveen A.L.
      • Palmblad M.
      • Falck D.
      • Wuhrer M.
      MassyTools: A high-throughput targeted data processing tool for relative quantitation and quality control developed for glycomic and glycoproteomic MALDI-MS.
      ,
      • Lynn K.S.
      • Chen C.C.
      • Lih T.M.
      • Cheng C.W.
      • Su W.C.
      • Chang C.H.
      • Cheng C.Y.
      • Hsu W.L.
      • Chen Y.J.
      • Sung T.Y.
      MAGIC: an automated N-linked glycoprotein identification tool using a Y1-ion pattern matching algorithm and in silico MS(2) approach.
      )
      Other (e.g. in-house program/script, manual annotation etc)(
      • Takakura D.
      • Harazono A.
      • Hashii N.
      • Kawasaki N.
      Selective glycopeptide profiling by acetone enrichment and LC/MS.
      ,
      • Chen R.
      • Seebun D.
      • Ye M.
      • Zou H.
      • Figeys D.
      Site-specific characterization of cell membrane N-glycosylation with integrated hydrophilic interaction chromatography solid phase extraction and LC-MS/MS.
      ,
      • Takakura D.
      • Tada M.
      • Kawasaki N.
      Membrane glycoproteomics of fetal lung fibroblasts using LC/MS.
      ,
      • Noro E.
      • Togayachi A.
      • Sato T.
      • Tomioka A.
      • Fujita M.
      • Sukegawa M.
      • Suzuki N.
      • Kaji H.
      • Narimatsu H.
      Large-scale identification of N-glycan glycoproteins carrying lewis x and site-specific N-glycan alterations in Fut9 Knockout mice.
      ,
      • Hong Q.
      • Ruhaak L.R.
      • Stroble C.
      • Parker E.
      • Huang J.
      • Maverakis E.
      • Lebrilla C.B.
      A method for comprehensive glycosite-mapping and direct quantitation of serum glycoproteins.
      ,
      • Ruiz-May E.
      • Hucko S.
      • Howe K.J.
      • Zhang S.
      • Sherwood R.W.
      • Thannhauser T.W.
      • Rose J.K.
      A comparative study of lectin affinity based plant N-glycoproteome profiling using tomato fruit as a model.
      ,
      • Goyallon A.
      • Cholet S.
      • Chapelle M.
      • Junot C.
      • Fenaille F.
      Evaluation of a combined glycomics and glycoproteomics approach for studying the major glycoproteins present in biofluids: Application to cerebrospinal fluid.
      ,
      • Scott N.E.
      • Marzook N.B.
      • Cain J.A.
      • Solis N.
      • Thaysen-Andersen M.
      • Djordjevic S.P.
      • Packer N.H.
      • Larsen M.R.
      • Cordwell S.J.
      Comparative proteomics and glycoproteomics reveal increased N-linked glycosylation and relaxed sequon specificity in Campylobacter jejuni NCTC11168 O.
      ,
      • Khatri K.
      • Staples G.O.
      • Leymarie N.
      • Leon D.R.
      • Turiák L.
      • Huang Y.
      • Yip S.
      • Hu H.
      • Heckendorf C.F.
      • Zaia J.
      Confident assignment of site-specific glycosylation in complex glycoproteins in a single step.
      )
      N-Glycome-assisted identification(
      • Thaysen-Andersen M.
      • Venkatakrishnan V.
      • Loke I.
      • Laurini C.
      • Diestel S.
      • Parker B.L.
      • Packer N.H.
      Human neutrophils secrete bioactive paucimannosidic proteins from azurophilic granules into pathogen-infected sputum.
      ,
      • Saraswat M.
      • Joenvaara S.
      • Musante L.
      • Peltoniemi H.
      • Holthofer H.
      • Renkonen R.
      N-linked (N-) glycoproteomics of urimary exosomes.
      ,
      • Parker B.L.
      • Thaysen-Andersen M.
      • Fazakerley D.J.
      • Holliday M.
      • Packer N.H.
      • James D.E.
      Terminal galactosylation and sialylation switching on membrane glycoproteins upon TNF-alpha-induced insulin resistance in adipocytes.
      ,
      • Parker B.L.
      • Thaysen-Andersen M.
      • Solis N.
      • Scott N.E.
      • Larsen M.R.
      • Graham M.E.
      • Packer N.H.
      • Cordwell S.J.
      Site-specific glycan-Peptide analysis for determination of N-glycoproteome heterogeneity.
      ,
      • Noro E.
      • Togayachi A.
      • Sato T.
      • Tomioka A.
      • Fujita M.
      • Sukegawa M.
      • Suzuki N.
      • Kaji H.
      • Narimatsu H.
      Large-scale identification of N-glycan glycoproteins carrying lewis x and site-specific N-glycan alterations in Fut9 Knockout mice.
      ,
      • Yang S.
      • Mishra S.
      • Chen L.
      • Zhou J.Y.
      • Chan D.W.
      • Chatterjee S.
      • Zhang H.
      Integrated glycoprotein immobilization method for glycopeptide and glycan analysis of cardiac hypertrophy.
      ,
      • Goyallon A.
      • Cholet S.
      • Chapelle M.
      • Junot C.
      • Fenaille F.
      Evaluation of a combined glycomics and glycoproteomics approach for studying the major glycoproteins present in biofluids: Application to cerebrospinal fluid.
      )
      Parallel proteome- and/or deglycoproteome (formerly occupied glycopeptides) profiling(
      • Thaysen-Andersen M.
      • Venkatakrishnan V.
      • Loke I.
      • Laurini C.
      • Diestel S.
      • Parker B.L.
      • Packer N.H.
      Human neutrophils secrete bioactive paucimannosidic proteins from azurophilic granules into pathogen-infected sputum.
      ,
      • Shah P.
      • Wang X.
      • Yang W.
      • Toghi Eshghi S.
      • Sun S.
      • Hoti N.
      • Chen L.
      • Yang S.
      • Pasay J.
      • Rubin A.
      • Zhang H.
      Integrated proteomic and glycoproteomic analyses of prostate cancer cells reveals glycoprotein alteration in protein abundance and glycosylation.
      ,
      • Saraswat M.
      • Joenvaara S.
      • Musante L.
      • Peltoniemi H.
      • Holthofer H.
      • Renkonen R.
      N-linked (N-) glycoproteomics of urimary exosomes.
      ,
      • Chen R.
      • Seebun D.
      • Ye M.
      • Zou H.
      • Figeys D.
      Site-specific characterization of cell membrane N-glycosylation with integrated hydrophilic interaction chromatography solid phase extraction and LC-MS/MS.
      ,
      • Takakura D.
      • Tada M.
      • Kawasaki N.
      Membrane glycoproteomics of fetal lung fibroblasts using LC/MS.
      ,
      • Parker B.L.
      • Thaysen-Andersen M.
      • Fazakerley D.J.
      • Holliday M.
      • Packer N.H.
      • James D.E.
      Terminal galactosylation and sialylation switching on membrane glycoproteins upon TNF-alpha-induced insulin resistance in adipocytes.
      ,
      • Parker B.L.
      • Thaysen-Andersen M.
      • Solis N.
      • Scott N.E.
      • Larsen M.R.
      • Graham M.E.
      • Packer N.H.
      • Cordwell S.J.
      Site-specific glycan-Peptide analysis for determination of N-glycoproteome heterogeneity.
      ,
      • Noro E.
      • Togayachi A.
      • Sato T.
      • Tomioka A.
      • Fujita M.
      • Sukegawa M.
      • Suzuki N.
      • Kaji H.
      • Narimatsu H.
      Large-scale identification of N-glycan glycoproteins carrying lewis x and site-specific N-glycan alterations in Fut9 Knockout mice.
      ,
      • Yang S.
      • Mishra S.
      • Chen L.
      • Zhou J.Y.
      • Chan D.W.
      • Chatterjee S.
      • Zhang H.
      Integrated glycoprotein immobilization method for glycopeptide and glycan analysis of cardiac hypertrophy.
      ,
      • Zhao Y.
      • Szeto S.S.
      • Kong R.P.
      • Law C.H.
      • Li G.
      • Quan Q.
      • Zhang Z.
      • Wang Y.
      • Chu I.K.
      Online two-dimensional porous graphitic carbon/reversed phase liquid chromatography platform applied to shotgun proteomics and glycoproteomics.
      ,
      • Liu M.
      • Zhang Y.
      • Chen Y.
      • Yan G.
      • Shen C.
      • Cao J.
      • Zhou X.
      • Liu X.
      • Zhang L.
      • Shen H.
      • Lu H.
      • He F.
      • Yang P.
      Efficient and accurate glycopeptide identification pipeline for high-throughput site-specific N-glycosylation analysis.
      ,
      • Goyallon A.
      • Cholet S.
      • Chapelle M.
      • Junot C.
      • Fenaille F.
      Evaluation of a combined glycomics and glycoproteomics approach for studying the major glycoproteins present in biofluids: Application to cerebrospinal fluid.
      )
      QuantitationSILAC(
      • Parker B.L.
      • Thaysen-Andersen M.
      • Fazakerley D.J.
      • Holliday M.
      • Packer N.H.
      • James D.E.
      Terminal galactosylation and sialylation switching on membrane glycoproteins upon TNF-alpha-induced insulin resistance in adipocytes.
      )
      iTRAQ(
      • Shah P.
      • Wang X.
      • Yang W.
      • Toghi Eshghi S.
      • Sun S.
      • Hoti N.
      • Chen L.
      • Yang S.
      • Pasay J.
      • Rubin A.
      • Zhang H.
      Integrated proteomic and glycoproteomic analyses of prostate cancer cells reveals glycoprotein alteration in protein abundance and glycosylation.
      ,
      • Yang S.
      • Mishra S.
      • Chen L.
      • Zhou J.Y.
      • Chan D.W.
      • Chatterjee S.
      • Zhang H.
      Integrated glycoprotein immobilization method for glycopeptide and glycan analysis of cardiac hypertrophy.
      )
      Other isotopes(
      • Kurogochi M.
      • Amano J.
      Relative quantitation of glycopeptides based on stable isotope labeling using MALDI-TOF MS.
      ,
      • Kim J.Y.
      • Oh D.
      • Kim S.K.
      • Kang D.
      • Moon M.H.
      Isotope-coded carbamidomethylation for quantification of N-glycoproteins with online microbore hollow fiber enzyme reactor-nanoflow liquid chromatography-tandem mass spectrometry.
      )
      Label-free quantitation (XIC, precursor ion intensity, spectral count, internal ref.)(
      • Thaysen-Andersen M.
      • Venkatakrishnan V.
      • Loke I.
      • Laurini C.
      • Diestel S.
      • Parker B.L.
      • Packer N.H.
      Human neutrophils secrete bioactive paucimannosidic proteins from azurophilic granules into pathogen-infected sputum.
      ,
      • Takakura D.
      • Harazono A.
      • Hashii N.
      • Kawasaki N.
      Selective glycopeptide profiling by acetone enrichment and LC/MS.
      ,
      • Kontro H.
      • Joenväärä S.
      • Haglund C.
      • Renkonen R.
      Comparison of sialylated N-glycopeptide levels in serum of pancreatic cancer patients, acute pancreatitis patients, and healthy controls.
      ,
      • Parker B.L.
      • Thaysen-Andersen M.
      • Solis N.
      • Scott N.E.
      • Larsen M.R.
      • Graham M.E.
      • Packer N.H.
      • Cordwell S.J.
      Site-specific glycan-Peptide analysis for determination of N-glycoproteome heterogeneity.
      ,
      • Liu M.
      • Zhang Y.
      • Chen Y.
      • Yan G.
      • Shen C.
      • Cao J.
      • Zhou X.
      • Liu X.
      • Zhang L.
      • Shen H.
      • Lu H.
      • He F.
      • Yang P.
      Efficient and accurate glycopeptide identification pipeline for high-throughput site-specific N-glycosylation analysis.
      ,
      • Mayampurath A.
      • Song E.
      • Mathur A.
      • Yu C.Y.
      • Hammoud Z.
      • Mechref Y.
      • Tang H.
      Label-free glycopeptide quantification for biomarker discovery in human sera.
      ,
      • Scott N.E.
      • Marzook N.B.
      • Cain J.A.
      • Solis N.
      • Thaysen-Andersen M.
      • Djordjevic S.P.
      • Packer N.H.
      • Larsen M.R.
      • Cordwell S.J.
      Comparative proteomics and glycoproteomics reveal increased N-linked glycosylation and relaxed sequon specificity in Campylobacter jejuni NCTC11168 O.
      )
      * Hydrazide is usually used to capture N-glycopeptides with a subsequent peptide N-glycosidase F release of formerly N-glycosylated peptides for glycosylation site mapping and is, thus, not a generally used tool in glycoproteomics.

      Enrichment and Prefractionation Strategies for Glycoproteomics

      Because of the substoichiometry of glycopeptides in complex peptide mixtures arising from the extensive glycan micro- and macro-heterogeneity, and inherently poor detectability, glycopeptide enrichment is a critical component of glycoproteomics experiments. Recent advances in glycopeptide enrichment have been fully reviewed elsewhere (
      • Song E.
      • Mechref Y.
      Defining glycoprotein cancer biomarkers by MS in conjunction with glycoprotein enrichment.
      ,
      • Scott N.E.
      • Cordwell S.J.
      Enrichment and identification of bacterial glycopeptides by mass spectrometry.
      ,
      • Chen C.C.
      • Su W.C.
      • Huang B.Y.
      • Chen Y.J.
      • Tai H.C.
      • Obena R.P.
      Interaction modes and approaches to glycopeptide and glycoprotein enrichment.
      ,
      • Zhao H.
      • Li Y.
      • Hu Y.
      Nanotechnologies in glycoproteomics.
      ). Exciting initiatives in glycopeptide enrichment strategies include the optimized use of boronic acid as a reversible glycopeptide capture method on magnetic graphene (
      • Wang J.
      • Wang Y.
      • Gao M.
      • Zhang X.
      • Yang P.
      Multilayer hydrophilic poly(phenol-formaldehyde resin)-coated magnetic graphene for boronic acid immobilization as a novel matrix for glycoproteome analysis.
      ) and on magnetic microspheres (
      • Wang M.
      • Zhang X.
      • Deng C.
      Facile synthesis of magnetic poly(styrene-co-4-vinylbenzene-boronic acid) microspheres for selective enrichment of glycopeptides.
      ). Boronic acid, which reacts with cis-diol-containing monosaccharide residues, has also recently been used to enrich intact glycopeptides (
      • Chen W.
      • Smeekens J.M.
      • Wu R.
      A universal chemical enrichment method for mapping the yeast N-glycoproteome by mass spectrometry (MS).
      ) and glycoproteins (
      • Xu Y.
      • Bailey U.M.
      • Punyadeera C.
      • Schulz B.L.
      Identification of salivary N-glycoproteins and measurement of glycosylation site occupancy by boronate glycoprotein enrichment and liquid chromatography/electrospray ionization tandem mass spectrometry.
      ), but remains an infrequently used glyco-enrichment method. Solid phase hydrazide-based glycopeptide capture has also seen developments (
      • Huang J.
      • Qin H.
      • Sun Z.
      • Huang G.
      • Mao J.
      • Cheng K.
      • Zhang Z.
      • Wan H.
      • Yao Y.
      • Dong J.
      • Zhu J.
      • Wang F.
      • Ye M.
      • Zou H.
      A peptide N-terminal protection strategy for comprehensive glycoproteome analysis using hydrazide chemistry based method.
      ) and applications (see Table I), but this approach is most commonly used in conjunction with peptide N-glycosidase F-catalyzed release and analysis of formerly N-glycosylated peptides and does not satisfy our definition of glycoproteomics. Other new enrichment methods of interest include the metabolic incorporation of N-azido sugars into N-glycopeptides to facilitate their specific enrichment and detection (
      • Woo C.M.
      • Iavarone A.T.
      • Spiciarich D.R.
      • Palaniappan K.K.
      • Bertozzi C.R.
      Isotope-targeted glycoproteomics (IsoTaG): a mass-independent platform for intact N- and O-glycopeptide discovery and analysis.
      ,
      • Smeekens J.M.
      • Chen W.
      • Wu R.
      Mass spectrometric analysis of the cell surface N-glycoproteome by combining metabolic labeling and click chemistry.
      ). The selective precipitation of glycopeptides by acetone (
      • Takakura D.
      • Harazono A.
      • Hashii N.
      • Kawasaki N.
      Selective glycopeptide profiling by acetone enrichment and LC/MS.
      ), use of size exclusion chromatography (
      • Saraswat M.
      • Joenväärä S.
      • Tomar A.K.
      • Singh S.
      • Yadav S.
      • Renkonen R.J.
      N-glycoproteomics of human seminal plasma glycoproteins.
      ,
      • Saraswat M.
      • Joenvaara S.
      • Musante L.
      • Peltoniemi H.
      • Holthofer H.
      • Renkonen R.
      N-linked (N-) glycoproteomics of urimary exosomes.
      ) and the combined use of porous graphitized carbon (PGC) and reversed phase (RP) (
      • Liu J.
      • Wang F.
      • Zhu J.
      • Mao J.
      • Liu Z.
      • Cheng K.
      • Qin H.
      • Zou H.
      Highly efficient N-glycoproteomic sample preparation by combining C(18) and graphitized carbon adsorbents.
      ) and titanium dioxide (
      • Melo-Braga M.N.
      • Schulz M.
      • Liu Q.
      • Swistowski A.
      • Palmisano G.
      • Engholm-Keller K.
      • Jakobsen L.
      • Zeng X.
      • Larsen M.R.
      Comprehensive quantitative comparison of the membrane proteome, phosphoproteome, and sialiome of human embryonic and neural stem cells.
      ) solid phase extraction (SPE) for efficient enrichment of glycopeptides and sialoglycopeptides, respectively, are also promising developments. However, common for most of these proof-of-principle methodology studies is the need for further validation to demonstrate their true potential in glycoproteomics. The frequently used zwitterionic-HILIC SPE-based methods for enrichment and analysis of intact N-glycopeptides (
      • Thaysen-Andersen M.
      • Thøgersen I.B.
      • Nielsen H.J.
      • Lademann U.
      • Brünner N.
      • Enghild J.J.
      • Højrup P.
      Rapid and individual-specific glycoprofiling of the low abundance N-glycosylated protein tissue inhibitor of metalloproteinases-1.
      ,
      • Mysling S.
      • Palmisano G.
      • Højrup P.
      • Thaysen-Andersen M.
      Utilizing ion-pairing hydrophilic interaction chromatography solid phase extraction for efficient glycopeptide enrichment in glycoproteomics.
      ,
      • Hägglund P.
      • Bunkenborg J.
      • Elortza F.
      • Jensen O.N.
      • Roepstorff P.
      A new strategy for identification of N-glycosylated proteins and unambiguous assignment of their glycosylation sites using HILIC enrichment and partial deglycosylation.
      ) have been further tested. Usefully, it was found that N-glycopeptides are still efficiently retained when using higher concentrations (0.1%) of surfactants and detergents including SDS and Triton X-100 (
      • Chen R.
      • Seebun D.
      • Ye M.
      • Zou H.
      • Figeys D.
      Site-specific characterization of cell membrane N-glycosylation with integrated hydrophilic interaction chromatography solid phase extraction and LC-MS/MS.
      ). In addition, other HILIC phases were used to enhance the loading capacity (
      • Dedvisitsakul P.
      • Jacobsen S.
      • Svensson B.
      • Bunkenborg J.
      • Finnie C.
      • Hägglund P.
      Glycopeptide enrichment using a combination of ZIC-HILIC and cotton wool for exploring the glycoproteome of wheat flour albumins.
      ) and have been synthetically tweaked by using “click-chemistry” (
      • Dedvisitsakul P.
      • Jacobsen S.
      • Svensson B.
      • Bunkenborg J.
      • Finnie C.
      • Hägglund P.
      Sample preparation for mass spectrometric analysis of human serum N-glycans using hydrophilic interaction chromatography-based solid phase extraction.
      ,
      • Cao L.
      • Yu L.
      • Guo Z.
      • Shen A.
      • Guo Y.
      • Liang X.
      N-Glycosylation site analysis of proteins from Saccharomyces cerevisiae by using hydrophilic interaction liquid chromatography-based enrichment, parallel deglycosylation, and mass spectrometry.
      ) and by introducing mixed mode-HILIC retention mechanisms (
      • Pan Y.
      • Ma C.
      • Tong W.
      • Fan C.
      • Zhang Q.
      • Zhang W.
      • Tian F.
      • Peng B.
      • Qin W.
      • Qian X.
      Preparation of sequence-controlled triblock copolymer-grafted silica microparticles by sequential-ATRP for highly efficient glycopeptides enrichment.
      ,
      • Huang G.
      • Xiong Z.
      • Qin H.
      • Zhu J.
      • Sun Z.
      • Zhang Y.
      • Peng X.
      • ou J.
      • Zou H.
      Synthesis of zwitterionic polymer brushes hybrid silica nanoparticles via controlled polymerization for highly efficient enrichment of glycopeptides.
      ,
      • Chen Y.
      • Xiong Z.
      • Zhang L.
      • Zhao J.
      • Zhang Q.
      • Peng L.
      • Zhang W.
      • Ye M.
      • Zou H.
      Facile synthesis of zwitterionic polymer-coated core-shell magnetic nanoparticles for highly specific capture of N-linked glycopeptides.
      ,
      • Bodnar E.D.
      • Perreault H.
      Synthesis and evaluation of carboxymethyl chitosan for glycopeptide enrichment.
      ,
      • Zheng J.
      • Xiao Y.
      • Wang L.
      • Lin Z.
      • Yang H.
      • Zhang L.
      • Chen G.
      Click synthesis of glucose-functionalized hydrophilic magnetic mesoporous nanoparticles for highly selective enrichment of glycopeptides and glycans.
      ). As all N-glycopeptides harbor a minimum degree of localized hydrophilicity arising from a high density of polar hydroxyl groups, HILIC remains the most used and, in our opinion, the most efficient and least biased enrichment method facilitating large-scale analysis of intact and native (nonderivatized) glycopeptides in N-glycoproteomics.
      Only few developments in the off-line separation and fractionation of glycopeptides prior to LC-MS/MS detection have recently been published. Some glycoproteomics approaches even by-pass this step because of the increased capacity of modern LC-MS/MS instrumentation to handle the extreme complexity of biologically-relevant glycopeptide mixtures, and perhaps because the multiple LC fractions resulting from off-line separations dramatically increase the required LC-MS/MS instrument time, lower the overall sensitivity of the analysis and complicate the downstream quantitative data analysis (
      • Takakura D.
      • Tada M.
      • Kawasaki N.
      Membrane glycoproteomics of fetal lung fibroblasts using LC/MS.
      ,
      • Kontro H.
      • Joenväärä S.
      • Haglund C.
      • Renkonen R.
      Comparison of sialylated N-glycopeptide levels in serum of pancreatic cancer patients, acute pancreatitis patients, and healthy controls.