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Glycoproteomic Analysis of Antibodies*

  • Gerhild Zauner
    Footnotes
    Affiliations
    Biomolecular Mass Spectrometry Unit, Postbus 9600, 2300RC Leiden University Medical Center, Leiden, The Netherlands
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  • Maurice H.J. Selman
    Footnotes
    Affiliations
    Biomolecular Mass Spectrometry Unit, Postbus 9600, 2300RC Leiden University Medical Center, Leiden, The Netherlands
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  • Albert Bondt
    Affiliations
    Biomolecular Mass Spectrometry Unit, Postbus 9600, 2300RC Leiden University Medical Center, Leiden, The Netherlands

    Department of Rheumatology, Erasmus University Medical Center, 3000CA Rotterdam, The Netherlands
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  • Yoann Rombouts
    Affiliations
    Department of Rheumatology, Leiden University Medical Center, Postbus 9600, 2300RC Leiden, The Netherlands
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  • Dennis Blank
    Affiliations
    Biomolecular Mass Spectrometry Unit, Postbus 9600, 2300RC Leiden University Medical Center, Leiden, The Netherlands
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  • André M. Deelder
    Affiliations
    Biomolecular Mass Spectrometry Unit, Postbus 9600, 2300RC Leiden University Medical Center, Leiden, The Netherlands
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  • Manfred Wuhrer
    Correspondence
    To whom correspondence should be addressed:Biomolecular Mass Spectrometry Unit, Department of Parasitology, Leiden University Medical Center, Postbus 9600, 2300RC Leiden, The Netherlands,
    Affiliations
    Biomolecular Mass Spectrometry Unit, Postbus 9600, 2300RC Leiden University Medical Center, Leiden, The Netherlands
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  • Author Footnotes
    * This work has been supported by funding from the European Union's Seventh Framework Programme (FP7-Health-F5-2011) under Grant Agreement No. 278535 (HighGlycan) and the Dutch Athritis foundation projects nr. NR 10-1-411 (for Albert Bondt) and NR 10-1-204 (for Yoann Rombouts). Maurice H. J. Selman thanks Hoffmann la Roche for financial support.
    This article contains supplemental material.
    § These authors contributed equally to this work.
Open AccessPublished:January 16, 2013DOI:https://doi.org/10.1074/mcp.R112.026005
      Antibody glycosylation has been shown to change with various processes. This review presents mass spectrometric approaches for antibody glycosylation analysis at the level of released glycans, glycopeptides, and intact protein. With regard to IgG fragment crystallizable glycosylation, mass spectrometry has shown its potential for subclass-specific, high-throughput analysis. In contrast, because of the vast heterogeneity of peptide moieties, fragment antigen binding glycosylation analysis of polyclonal IgG relies entirely on glycan release. Next to IgG, IgA has gained some attention, and studies of its O- and N-glycosylation have revealed disease-associated glycosylation changes. Glycoproteomic analyses of IgM and IgE are lagging behind but should complete our picture of glycosylation's influence on antibody function.

      BIOLOGICAL ROLE OF IMMUNOGLOBULIN GLYCOSYLATION

      Immunoglobulins (Igs)
      The abbreviations used are:
      ECD
      electron capture dissociation
      ESI
      electrospray ionization
      Fab
      fragment antigen binding
      Fc
      fragment crystallizable
      FTICR
      Fourier transform ion cyclotron resonance
      Gal
      galactoses
      GlcNAc
      N-acetylglucosamine
      HILIC
      hydrophilic interaction liquid chromatography
      HR
      hinge region
      Ig
      immunglobulin
      mAb
      monoclonal antibody
      RP
      reversed-phase.
      are produced by the adaptive immune system in order to identify and neutralize foreign antigens and pathogens to which the host has been exposed. In humans, five known classes of Igs (IgG, IgM, IgA, IgE, and IgD) are secreted in variable amounts by B cells during an immune response. Although these Ig classes are built from Ig domains and are thus structurally related, they differ considerably in several aspects, such as their glycosylation (
      • Arnold J.N.
      • Wormald M.R.
      • Sim R.B.
      • Rudd P.M.
      • Dwek R.A.
      The impact of glycosylation on the biological function and structure of human immunoglobulins.
      ). Over the past 30 years, numerous studies have explored the structural, biological, and clinical roles of Ig glycosylation, focusing mainly on IgG molecules, which are the most abundant serum Ig, occurring at 10 to 15 mg/ml (value for IgG1) in human circulation (
      • Arnold J.N.
      • Wormald M.R.
      • Sim R.B.
      • Rudd P.M.
      • Dwek R.A.
      The impact of glycosylation on the biological function and structure of human immunoglobulins.
      ). Each IgG molecule consists of two heavy and two light chains that together form two fragment antigen binding (Fab) portions and one fragment crystallizable (Fc) portion (Fig. 1). Two N-glycans are linked to the heavy chains at Asn 297 in the CH2 domain of the protein backbone (Fc part). These Fc glycans are in part located in a cavity between the two heavy chains and influence the conformation of the protein (
      • Borrok M.J.
      • Jung S.T.
      • Kang T.H.
      • Monzingo A.F.
      • Georgiou G.
      Revisiting the role of glycosylation in the structure of human IgG fc.
      ,
      • Krapp S.
      • Mimura Y.
      • Jefferis R.
      • Huber R.
      • Sondermann P.
      Structural analysis of human IgG-Fc glycoforms reveals a correlation between glycosylation and structural integrity.
      ). Their removal by glycosidases or via mutation of the glycosylation sites reduces the binding of IgG to Fc-gamma receptors (FcγR) (
      • Jung S.T.
      • Reddy S.T.
      • Kang T.H.
      • Borrok M.J.
      • Sandlie I.
      • Tucker P.W.
      • Georgiou G.
      Aglycosylated IgG variants expressed in bacteria that selectively bind FcgammaRI potentiate tumor cell killing by monocyte-dendritic cells.
      ,
      • Nesspor T.C.
      • Raju T.S.
      • Chin C.N.
      • Vafa O.
      • Brezski R.J.
      Avidity confers FcgammaR binding and immune effector function to aglycosylated immunoglobulin G1.
      ,
      • Tao M.H.
      • Morrison S.L.
      Studies of aglycosylated chimeric mouse-human IgG. Role of carbohydrate in the structure and effector functions mediated by the human IgG constant region.
      ). The Fc-linked carbohydrates are complex-type biantennary N-glycans with a high level of core-fucosylation and a variable number of galactoses (Gal) resulting in the prevalent glycoforms G0F (no Gal), G1F (one Gal), and G2F (two Gal). A minor proportion of these glycans might contain a bisecting N-acetylglucosamine (GlcNAc) residue and/or terminal sialic acids substituting antenna Gal (
      • Parekh R.B.
      • Dwek R.A.
      • Sutton B.J.
      • Fernandes D.L.
      • Leung A.
      • Stanworth D.
      • Rademacher T.W.
      • Mizuochi T.
      • Taniguchi T.
      • Matsuta K.
      • Takeuchi F.
      • Nagano Y.
      • Miyamoto T.
      • Kobata A.
      Association of rheumatoid arthritis and primary osteoarthritis with changes in the glycosylation pattern of total serum IgG.
      ) (see Fig. 1).
      Figure thumbnail gr1
      Fig. 1Glycoproteomic analysis of human IgG and IgA. Glycosylation of IgG1 (P01857), IgG2 (P01859), IgG3 (P01860), IgG4 (P01861), IgA1 (P01876) in secretory IgA (sIgA), and IgA2 (P01877). Heavy chains are depicted in gray, light chains in blue, secretory components (P01833) of sIgA in purple, and joining chain (P01591) in orange. Glycosylation sites are indicated by the respective amino acid number and schematic N- and O-glycans, respectively. Tryptic peptides for all constant region glycosylation sites are given except for the secretory component and the joining chain of sIgA. Glycans are depicted according to CFG notation; blue square, N-acetylglucosamine; green circle, mannose; yellow circle, galactose; red triangle, fucose; purple diamond, sialic acid. If known, further information on N-glycan structures is given (
      • Selman M.H.
      • Derks R.J.
      • Bondt A.
      • Palmblad M.
      • Schoenmaker B.
      • Koeleman C.A.
      • van de Geijn F.E.
      • Dolhain R.J.
      • Deelder A.M.
      • Wuhrer M.
      Fc specific IgG glycosylation profiling by robust nano-reverse phase HPLC-MS using a sheath-flow ESI sprayer interface.
      ,
      • Anumula K.R.
      Quantitative glycan profiling of normal human plasma derived immunoglobulin and its fragments Fab and Fc.
      ,
      • Arnold J.N.
      • Radcliffe C.M.
      • Wormald M.R.
      • Royle L.
      • Harvey D.J.
      • Crispin M.
      • Dwek R.A.
      • Sim R.B.
      • Rudd P.M.
      The glycosylation of human serum IgD and IgE and the accessibility of identified oligomannose structures for interaction with mannan-binding lectin.
      ). For IgA, values in parentheses indicate the abundance in plasma IgA/sIgA. The composition of O-glycans on the IgA HR peptide is reported according to Deshpande et al. (
      • Deshpande N.
      • Jensen P.H.
      • Packer N.H.
      • Kolarich D.
      GlycoSpectrumScan: fishing glycopeptides from MS spectra of protease digests of human colostrum sIgA.
      ).
      Many reports have described variations of IgG Fc glycosylation, especially of the degree of galactosylation, related to age, sex, heritability, and pregnancy, as well as to autoimmune diseases, infectious diseases, and cancers (e.g. Refs.
      • Parekh R.
      • Isenberg D.
      • Rook G.
      • Roitt I.
      • Dwek R.
      • Rademacher T.
      A comparative analysis of disease-associated changes in the galactosylation of serum IgG.
      ,
      • Ercan A.
      • Barnes M.G.
      • Hazen M.
      • Tory H.
      • Henderson L.
      • Dedeoglu F.
      • Fuhlbrigge R.C.
      • Grom A.
      • Holm I.A.
      • Kellogg M.
      • Kim S.
      • Adamczyk B.
      • Rudd P.M.
      • Son M.B.
      • Sundel R.P.
      • Foell D.
      • Glass D.N.
      • Thompson S.D.
      • Nigrovic P.A.
      Multiple juvenile idiopathic arthritis subtypes demonstrate proinflammatory IgG glycosylation.
      ,
      • Pucic M.
      • Knezevic A.
      • Vidic J.
      • Adamczyk B.
      • Novokmet M.
      • Polasek O.
      • Gornik O.
      • Supraha-Goreta S.
      • Wormald M.R.
      • Redzic I.
      • Campbell H.
      • Wright A.
      • Hastie N.D.
      • Wilson J.F.
      • Rudan I.
      • Wuhrer M.
      • Rudd P.M.
      • Josic D.
      • Lauc G.
      High throughput isolation and glycosylation analysis of IgG-variability and heritability of the IgG glycome in three isolated human populations.
      ,
      • Ruhaak L.R.
      • Uh H.W.
      • Beekman M.
      • Koeleman C.A.
      • Hokke C.H.
      • Westendorp R.G.
      • Wuhrer M.
      • Houwing-Duistermaat J.J.
      • Slagboom P.E.
      • Deelder A.M.
      Decreased levels of bisecting GlcNAc glycoforms of IgG are associated with human longevity.
      ,
      • Saldova R.
      • Royle L.
      • Radcliffe C.M.
      • Abd Hamid U.M.
      • Evans R.
      • Arnold J.N.
      • Banks R.E.
      • Hutson R.
      • Harvey D.J.
      • Antrobus R.
      • Petrescu S.M.
      • Dwek R.A.
      • Rudd P.M.
      Ovarian cancer is associated with changes in glycosylation in both acute-phase proteins and IgG.
      ,
      • van de Geijn F.E.
      • Wuhrer M.
      • Selman M.H.
      • Willemsen S.P.
      • de Man Y.A.
      • Deelder A.M.
      • Hazes J.M.
      • Dolhain R.J.
      Immunoglobulin G galactosylation and sialylation are associated with pregnancy-induced improvement of rheumatoid arthritis and the postpartum flare: results from a large prospective cohort study.
      ,
      • Bones J.
      • Mittermayr S.
      • O'Donoghue N.
      • Guttman A.
      • Rudd P.M.
      Ultra performance liquid chromatographic profiling of serum N-glycans for fast and efficient identification of cancer associated alterations in glycosylation.
      ,
      • Huhn C.
      • Selman M.H.
      • Ruhaak L.R.
      • Deelder A.M.
      • Wuhrer M.
      IgG glycosylation analysis.
      ). For instance, an increase in IgG G0F is observed in the serum of patients with rheumatoid arthritis (
      • Parekh R.B.
      • Dwek R.A.
      • Sutton B.J.
      • Fernandes D.L.
      • Leung A.
      • Stanworth D.
      • Rademacher T.W.
      • Mizuochi T.
      • Taniguchi T.
      • Matsuta K.
      • Takeuchi F.
      • Nagano Y.
      • Miyamoto T.
      • Kobata A.
      Association of rheumatoid arthritis and primary osteoarthritis with changes in the glycosylation pattern of total serum IgG.
      ) and correlates with disease progression and severity (
      • Gindzienska-Sieskiewicz E.
      • Klimiuk P.A.
      • Kisiel D.G.
      • Gindzienski A.
      • Sierakowski S.
      The changes in monosaccharide composition of immunoglobulin G in the course of rheumatoid arthritis.
      ,
      • van Zeben D.
      • Rook G.A.
      • Hazes J.M.
      • Zwinderman A.H.
      • Zhang Y.
      • Ghelani S.
      • Rademacher T.W.
      • Breedveld F.C.
      Early agalactosylation of IgG is associated with a more progressive disease course in patients with rheumatoid arthritis: results of a follow-up study.
      ). These clinical observations have led researchers to examine in detail the relationship between Fc glycan structures, the biological properties of IgG, and the degree of inflammation. It was found that an absence of sialic acids and low levels of galactosylation might confer important pro-inflammatory properties to IgG by facilitating the formation of immune complexes and favoring the binding of IgG to activating FcγR (
      • Jefferis R.
      • Lund J.
      • Pound J.D.
      IgG-Fc-mediated effector functions: molecular definition of interaction sites for effector ligands and the role of glycosylation.
      ,
      • Kaneko Y.
      • Nimmerjahn F.
      • Ravetch J.V.
      Anti-inflammatory activity of immunoglobulin G resulting from Fc sialylation.
      ,
      • Nimmerjahn F.
      • Anthony R.M.
      • Ravetch J.V.
      Agalactosylated IgG antibodies depend on cellular Fc receptors for in vivo activity.
      ). Similarly, the absence of core-fucose or the presence of bisecting GlcNAc improved the affinity of the Fc tail to FcγRIIIa, thereby enhancing antibody-dependent cellular cytotoxicity (
      • Shields R.L.
      • Lai J.
      • Keck R.
      • O'Connell L.Y.
      • Hong K.
      • Meng Y.G.
      • Weikert S.H.
      • Presta L.G.
      Lack of fucose on human IgG1 N-linked oligosaccharide improves binding to human Fcgamma RIII and antibody-dependent cellular toxicity.
      ,
      • Ferrara C.
      • Grau S.
      • Jager C.
      • Sondermann P.
      • Brunker P.
      • Waldhauer I.
      • Hennig M.
      • Ruf A.
      • Rufer A.C.
      • Stihle M.
      • Umana P.
      • Benz J.
      Unique carbohydrate-carbohydrate interactions are required for high affinity binding between FcgammaRIII and antibodies lacking core fucose.
      ,
      • Zou G.
      • Ochiai H.
      • Huang W.
      • Yang Q.
      • Li C.
      • Wang L.X.
      Chemoenzymatic synthesis and Fcgamma receptor binding of homogeneous glycoforms of antibody Fc domain. Presence of a bisecting sugar moiety enhances the affinity of Fc to FcgammaIIIa receptor.
      ). On this basis, new glycoengineered anti-cancer antibodies carrying afucosylated Fc glycans are currently in clinical development, such as the anti-CD20 monoclonal antibody (mAb) obinutuzumab (GA101) for use against B-cell lymphoma (
      • Salles G.
      • Morschhauser F.
      • Lamy T.
      • Milpied N.
      • Thieblemont C.
      • Tilly H.
      • Bieska G.
      • Asikanius E.
      • Carlile D.
      • Birkett J.
      • Pisa P.
      • Cartron G.
      Phase 1 study results of the type II glycoengineered humanized anti-CD20 monoclonal antibody obinutuzumab (GA101) in B-cell lymphoma patients.
      ,
      • Sehn L.H.
      • Assouline S.E.
      • Stewart D.A.
      • Mangel J.
      • Gascoyne R.D.
      • Fine G.
      • Frances-Lasserre S.
      • Carlile D.J.
      • Crump M.
      A phase 1 study of obinutuzumab induction followed by 2 years of maintenance in patients with relapsed CD20-positive B-cell malignancies.
      ).
      In addition, Fc-linked glycans appear to modulate the activation of the complement system. Whereas the classical complement pathway can be triggered by the preferential binding of C1q to fully galactosylated IgG, the lectin pathway is recruited through the recognition of agalactosylated IgG by mannose-binding lectin (
      • Malhotra R.
      • Wormald M.R.
      • Rudd P.M.
      • Fischer P.B.
      • Dwek R.A.
      • Sim R.B.
      Glycosylation changes of IgG associated with rheumatoid arthritis can activate complement via the mannose-binding protein.
      ,
      • Raju T.S.
      Terminal sugars of Fc glycans influence antibody effector functions of IgGs.
      ). In contrast, the presence of terminal galactose and/or sialic acid residues on Fc glycans might confer anti-inflammatory properties to IgG via interaction with the human lectins Dectin-1 (
      • Karsten C.M.
      • Pandey M.K.
      • Figge J.
      • Kilchenstein R.
      • Taylor P.R.
      • Rosas M.
      • McDonald J.U.
      • Orr S.J.
      • Berger M.
      • Petzold D.
      • Blanchard V.
      • Winkler A.
      • Hess C.
      • Reid D.M.
      • Majoul I.V.
      • Strait R.T.
      • Harris N.L.
      • Kohl G.
      • Wex E.
      • Ludwig R.
      • Zillikens D.
      • Nimmerjahn F.
      • Finkelman F.D.
      • Brown G.D.
      • Ehlers M.
      • Kohl J.
      Anti-inflammatory activity of IgG1 mediated by Fc galactosylation and association of FcgammaRIIB and dectin-1.
      ) and dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (
      • Kaneko Y.
      • Nimmerjahn F.
      • Ravetch J.V.
      Anti-inflammatory activity of immunoglobulin G resulting from Fc sialylation.
      ,
      • Anthony R.M.
      • Ravetch J.V.
      A novel role for the IgG Fc glycan: the anti-inflammatory activity of sialylated IgG Fcs.
      ,
      • Anthony R.M.
      • Kobayashi T.
      • Wermeling F.
      • Ravetch J.V.
      Intravenous gammaglobulin suppresses inflammation through a novel T(H)2 pathway.
      ). Thus, variations in the structure of IgG Fc glycans might skew the immune system toward a pro- or an anti-inflammatory response by modulating the interaction of IgG with several immune components, including FcγR, complement factors, and lectins. Interestingly, it was recently established that IgG Fc glycosylation may be modulated by factors such as hormones (e.g. estradiol and progesterone), cytokines (e.g. IFN-γ and IL-21), bacterial DNA (CpG oligodeoxynucleotide), and food metabolites (e.g. all-trans retinoic acid and drugs) (
      • Chen G.
      • Wang Y.
      • Qiu L.
      • Qin X.
      • Liu H.
      • Wang X.
      • Wang Y.
      • Song G.
      • Li F.
      • Guo Y.
      • Li F.
      • Guo S.
      • Li Z.
      Human IgG Fc-glycosylation profiling reveals associations with age, sex, female sex hormones and thyroid cancer.
      ,
      • Prados M.B.
      • La B.J.
      • Szekeres-Bartho J.
      • Caramelo J.
      • Miranda S.
      Progesterone induces a switch in oligosaccharyltransferase isoform expression: consequences on IgG N-glycosylation.
      ,
      • Wang J.
      • Balog C.I.
      • Stavenhagen K.
      • Koeleman C.A.
      • Scherer H.U.
      • Selman M.H.
      • Deelder A.M.
      • Huizinga T.W.
      • Toes R.E.
      • Wuhrer M.
      Fc-glycosylation of IgG1 is modulated by B-cell stimuli.
      ).
      The influence of glycosylation on the biological properties of other Ig classes has been poorly explored. Some reports have established that variations in the glycosylation of IgA and IgE modulate the affinity for their respective receptors, FcαR and FcεR (
      • Arnold J.N.
      • Wormald M.R.
      • Sim R.B.
      • Rudd P.M.
      • Dwek R.A.
      The impact of glycosylation on the biological function and structure of human immunoglobulins.
      ). Results from clinical studies also support the idea that there is some structural and functional role of glycosylation in all classes of Ig. An example is IgA1, which exhibits O-glycosylation at various sites of its hinge region peptide (see Fig. 1). In nephropathy, lowered levels of IgA1 O-glycan sialylation and galactosylation have been observed (
      • Novak J.
      • Julian B.A.
      • Tomana M.
      • Mestecky J.
      IgA glycosylation and IgA immune complexes in the pathogenesis of IgA nephropathy.
      ). These abnormally glycosylated IgA1s were shown to have a longer half-life, to self-aggregate, and to form complexes with other molecules of the immune system, including IgG and mannose-binding lectin, thereby promoting IgA deposition in the kidney mesangium and exacerbating inflammation (
      • Arnold J.N.
      • Wormald M.R.
      • Sim R.B.
      • Rudd P.M.
      • Dwek R.A.
      The impact of glycosylation on the biological function and structure of human immunoglobulins.
      ).

      ANALYTICAL APPROACHES FOR Ig GLYCOSYLATION ANALYSIS

      Glycosylation analysis of glycoproteins in general and of Igs in particular may be addressed via (a) intact glycoprotein analysis, (b) the characterization of glycopeptides, or (c) structural analysis of chemically or enzymatically released glycans (
      • Huhn C.
      • Selman M.H.
      • Ruhaak L.R.
      • Deelder A.M.
      • Wuhrer M.
      IgG glycosylation analysis.
      ,
      • Dalpathado D.S.
      • Desaire H.
      Glycopeptide analysis by mass spectrometry.
      ,
      • Morelle W.
      • Michalski J.C.
      Analysis of protein glycosylation by mass spectrometry.
      ). Mass spectrometric analysis of glycoproteins at the glycopeptide or released glycan level are currently the methods of choice for obtaining sensitive and comprehensive glycosylation information from complex biological samples (
      • Kolarich D.
      • Jensen P.H.
      • Altmann F.
      • Packer N.H.
      Determination of site-specific glycan heterogeneity on glycoproteins.
      ).
      Analysis at the glycopeptide level is the most favorable approach, as site-specific glycan heterogeneity can be characterized and glycan compositions can be correlated to their attachment sites on the protein (
      • Dalpathado D.S.
      • Desaire H.
      Glycopeptide analysis by mass spectrometry.
      ). In particular, liquid chromatography–mass spectrometry (LC/MS) has been widely used for glycopeptide analysis. The advantage of LC-electrospray ionization (ESI)-MS analysis is the up-front chromatographic separation of the (glyco)peptides prior to MS analysis. Obviously the choice of an efficient chromatographic separation method for a glycopeptide mixture after proteolytic digestion is crucial. For this purpose, C18 reversed-phase (RP) HPLC is widely used, next to hydrophilic interaction liquid chromatography (HILIC) and graphitized carbon HPLC (
      • Huhn C.
      • Selman M.H.
      • Ruhaak L.R.
      • Deelder A.M.
      • Wuhrer M.
      IgG glycosylation analysis.
      ,
      • Zauner G.
      • Deelder A.M.
      • Wuhrer M.
      Recent advances in hydrophilic interaction liquid chromatography (HILIC) for structural glycomics.
      ,
      • Nwosu C.C.
      • Seipert R.R.
      • Strum J.S.
      • Hua S.S.
      • An H.J.
      • Zivkovic A.M.
      • German B.J.
      • Lebrilla C.B.
      Simultaneous and extensive site-specific N- and O-glycosylation analysis in protein mixtures.
      ).
      Released glycan samples are generally of lower complexity than glycopeptide samples, and various targeted and untargeted glycomics approaches are commonly applied at the released glycan level. A common high-throughput approach involves the permethylation of C18 RP and carbon solid phase extraction purified glycans followed by matrix-assisted lased desorption ionization (MALDI) time-of-flight (TOF) MS analysis (
      • Morelle W.
      • Michalski J.C.
      Analysis of protein glycosylation by mass spectrometry.
      ,
      • Mechref Y.
      • Hu Y.
      • Garcia A.
      • Zhou S.
      • Desantos-Garcia J.L.
      • Hussein A.
      Defining putative glycan cancer biomarkers by MS.
      ). This review focuses mainly on Ig glycosylation analysis via MS of glycopeptides, and we refer to other reviews for more in-depth coverage of released glycan analysis (
      • Mechref Y.
      • Muzikar J.
      • Novotny M.V.
      Comprehensive assessment of N-glycans derived from a murine monoclonal antibody: a case for multimethodological approach.
      ,
      • Ruhaak L.R.
      • Zauner G.
      • Huhn C.
      • Bruggink C.
      • Deelder A.M.
      • Wuhrer M.
      Glycan labeling strategies and their use in identification and quantification.
      ,
      • Harvey D.J.
      Analysis of carbohydrates and glycoconjugates by matrix-assisted laser desorption/ionization mass spectrometry: an update for 2007–2008.
      ,
      • Leymarie N.
      • Zaia J.
      Effective use of mass spectrometry for glycan and glycopeptide structural analysis.
      ).

      Total IgG Glycosylation Analysis

      Polyclonal human IgG N-glycosylation has been studied extensively at the level of released N-glycans. A seminal 1985 work by Parekh et al. demonstrated increased levels of agalactosylated glycans associated with rheumatoid arthritis and osteoarthritis (
      • Parekh R.B.
      • Dwek R.A.
      • Sutton B.J.
      • Fernandes D.L.
      • Leung A.
      • Stanworth D.
      • Rademacher T.W.
      • Mizuochi T.
      • Taniguchi T.
      • Matsuta K.
      • Takeuchi F.
      • Nagano Y.
      • Miyamoto T.
      • Kobata A.
      Association of rheumatoid arthritis and primary osteoarthritis with changes in the glycosylation pattern of total serum IgG.
      ). That paper represents a major milestone in IgG research and gave rise to a continuing range of studies on human IgG glycosylation using a diverse range of methods for glycan analysis such as HILIC with fluorescence detection, capillary gel electrophoresis with laser-induced fluorescence detection, and MS, demonstrating IgG glycosylation changes with age, sex, pregnancy, and diseases (
      • Bones J.
      • Mittermayr S.
      • O'Donoghue N.
      • Guttman A.
      • Rudd P.M.
      Ultra performance liquid chromatographic profiling of serum N-glycans for fast and efficient identification of cancer associated alterations in glycosylation.
      ,
      • Huhn C.
      • Selman M.H.
      • Ruhaak L.R.
      • Deelder A.M.
      • Wuhrer M.
      IgG glycosylation analysis.
      ).
      A high-throughput isolation and glycosylation analysis of IgG was published recently by Pucic et al. (
      • Pucic M.
      • Knezevic A.
      • Vidic J.
      • Adamczyk B.
      • Novokmet M.
      • Polasek O.
      • Gornik O.
      • Supraha-Goreta S.
      • Wormald M.R.
      • Redzic I.
      • Campbell H.
      • Wright A.
      • Hastie N.D.
      • Wilson J.F.
      • Rudan I.
      • Wuhrer M.
      • Rudd P.M.
      • Josic D.
      • Lauc G.
      High throughput isolation and glycosylation analysis of IgG-variability and heritability of the IgG glycome in three isolated human populations.
      ): IgGs of 2298 individuals were efficiently isolated from plasma using a 96-well protein G monolithic plate. The N-glycans were released using PNGase F, labeled with 2-aminobenzamide, and analyzed by means of HILIC HPLC with fluorescence detection. High variability in IgG glycosylation among individuals was observed and was found to be approximately three times higher than in the total plasma N-glycome. Heritability in this case was found to be between 30% and 50%, and gender appeared not to be an important predictor for any IgG glycans. Sialylation was found to be the most endogenously defined glycosylation feature, with up to 60% of variance explained by heritability.
      Analysis of total IgG glycosylation at the level of released glycans registers mixtures of Fc and Fab glycans from the different subclasses of IgG. Approaches that provide more specific information on IgG glycosylation are presented in the following two sections.

      IgG Fc Glycosylation Analysis

      Mass spectrometric analysis of tryptic Fc glycopeptides allows the discrimination of different human IgG subclasses based on minor differences in amino acid sequences (Fig. 2). For IgG3, different allotypes appear to prevail in different ethnic groups (
      • Dard P.
      • Lefranc M.P.
      • Osipova L.
      • Sanchez-Mazas A.
      DNA sequence variability of IGHG3 alleles associated to the main G3m haplotypes in human populations.
      ,
      • Jefferis R.
      • Lefranc M.P.
      Human immunoglobulin allotypes: possible implications for immunogenicity.
      ). Analysis of IgG3 from Caucasians mainly revealed allotype G3m(b*), which exhibits a phenylalanine (F) in position 296 (
      • Balbin M.
      • Grubb A.
      • de Lange G.G.
      • Grubb R.
      DNA sequences specific for Caucasian G3m(b) and(g) allotypes: allotyping at the genomic level.
      ). As a consequence, the resulting tryptic Fc glycopeptides of IgG2 and IgG3 show identical peptide moieties. In contrast, IgG3 from Asian donors was reported to exhibit a tyrosine (Y) in position 296, resulting in identical peptide moieties for IgG3 and IgG4 (
      • Dard P.
      • Lefranc M.P.
      • Osipova L.
      • Sanchez-Mazas A.
      DNA sequence variability of IGHG3 alleles associated to the main G3m haplotypes in human populations.
      ). Thus, allotypic variations have to be taken into account when comparing subclass-specific IgG Fc-glycosylation profiles of genetically different groups.
      Figure thumbnail gr2
      Fig. 2Murine and human plasma IgG Fc glycosylation differences. Tryptic glycopeptides (A) of murine (IgG1, BAE25911, BAC30871; IgG2a, P01863, P01864; IgG2b, P01867; IgG3, P03987) and human () polyclonal IgG were analyzed via RP-nano-LC sheath-flow ESI-MS using a gradient of aqueous 0.1% trifluoroacetic acid and acetonitrile. Spectra represent the sum of a 45-s elution window depicting [M+3H]3+ species of murine IgG2a/b (B) and human IgG1 (C) Fc glycoforms.
      A very convenient approach for IgG Fc glycosylation analysis is the measurement of (tryptic) Fc glycopeptides, which is generally performed via RP-LC-MS/MS (
      • Kolarich D.
      • Jensen P.H.
      • Altmann F.
      • Packer N.H.
      Determination of site-specific glycan heterogeneity on glycoproteins.
      ,
      • Perdivara I.
      • Deterding L.J.
      • Cozma C.
      • Tomer K.B.
      • Przybylski M.
      Glycosylation profiles of epitope-specific anti-beta-amyloid antibodies revealed by liquid chromatography-mass spectrometry.
      ,
      • Stadlmann J.
      • Pabst M.
      • Kolarich D.
      • Kunert R.
      • Altmann F.
      Analysis of immunoglobulin glycosylation by LC-ESI-MS of glycopeptides and oligosaccharides.
      ,
      • Wuhrer M.
      • Stam J.C.
      • van de Geijn F.E.
      • Koeleman C.A.
      • Verrips C.T.
      • Dolhain R.J.
      • Hokke C.H.
      • Deelder A.M.
      Glycosylation profiling of immunoglobulin G (IgG) subclasses from human serum.
      ). Chromatographic separation is observed on the basis of small structural differences in a single amino acid side chain. Tryptic Fc glycopeptides of IgG1 carrying tyrosine residues in positions 296 and 300 elute in front of tryptic IgG3/4 glycopeptides (F296 and Y300), which again elute in front of tryptic IgG2/3 Fc glycopeptides (F296 and F300). In contrast, changes in the glycan structure with regard to galactosylation, fucosylation, and bisection hardly affect RP retention times. Consequently, IgG1, IgG2/3, and IgG3/4 glycopeptide clusters are observed in distinct retention time windows. Isomeric tryptic Fc glycopeptide species belonging to different IgG subclasses (i.e. fucosylated IgG1 and non-fucosylated IgG3/4, or fucosylated IgG3/4 and non-fucosylated IgG2/3) are consistently separated by RP-LC, allowing their unambiguous assignment to specific IgG subclasses upon mass spectrometric detection. Sialic acid, however, can have a strong influence on IgG Fc glycopeptide retention, depending on the solvent system. The use of an acetonitrile gradient in aqueous 0.1% formic acid results in greater retention of sialylated species than neutral glycopeptides (
      • Perdivara I.
      • Deterding L.J.
      • Cozma C.
      • Tomer K.B.
      • Przybylski M.
      Glycosylation profiles of epitope-specific anti-beta-amyloid antibodies revealed by liquid chromatography-mass spectrometry.
      ,
      • Wuhrer M.
      • Stam J.C.
      • van de Geijn F.E.
      • Koeleman C.A.
      • Verrips C.T.
      • Dolhain R.J.
      • Hokke C.H.
      • Deelder A.M.
      Glycosylation profiling of immunoglobulin G (IgG) subclasses from human serum.
      ). We recently optimized an RP-nano-LC-ESI-MS setup for fast and robust subclass-specific Fc-glycosylation profiling in large sets of IgG samples (
      • Selman M.H.
      • Derks R.J.
      • Bondt A.
      • Palmblad M.
      • Schoenmaker B.
      • Koeleman C.A.
      • van de Geijn F.E.
      • Dolhain R.J.
      • Deelder A.M.
      • Wuhrer M.
      Fc specific IgG glycosylation profiling by robust nano-reverse phase HPLC-MS using a sheath-flow ESI sprayer interface.
      ). Tryptic (glyco)peptides of protein A or protein G affinity-purified polyclonal IgG were collected onto a porous particle C18 trap column and separated on a fused-core C18 column using a short gradient of aqueous 0.1% trifluoroacetic acid and acetonitrile (16 min total analysis time). When formic acid was replaced with trifluoracetic acid, sialylated glycopeptides eluted together with their non-sialylated counterparts. Long-term stable and robust mass spectrometric analysis was achieved by employing a sheath-flow ESI sprayer with isopropanol:water:propionic acid (50:30:20; v:v:v) as a sheath liquid. The relative standard deviations for the eight major observed glycopeptide species remained less than 4% over a time range of several months, thereby allowing the analysis of thousands of samples with high precision.
      HILIC-LC-MS also has been reported as a versatile tool for the separation of glycans and glycopeptides (
      • Gilar M.
      • Yu Y.Q.
      • Ahn J.
      • Xie H.
      • Han H.
      • Ying W.
      • Qian X.
      Characterization of glycoprotein digests with hydrophilic interaction chromatography and mass spectrometry.
      ,
      • Singh C.
      • Zampronio C.G.
      • Creese A.J.
      • Cooper H.J.
      Higher energy collision dissociation (HCD) product ion-triggered electron transfer dissociation (ETD) mass spectrometry for the analysis of N-linked glycoproteins.
      ,
      • Takegawa Y.
      • Deguchi K.
      • Keira T.
      • Ito H.
      • Nakagawa H.
      • Nishimura S.
      Separation of isomeric 2-aminopyridine derivatized N-glycans and N-glycopeptides of human serum immunoglobulin G by using a zwitterionic type of hydrophilic-interaction chromatography.
      ). Tryptic IgG1 Fc glycopeptides experience more retention than IgG2 Fc glycopeptides as a result of the additional oxygen atoms presented by the tyrosine residues at positions 296 and 300 (
      • Takegawa Y.
      • Deguchi K.
      • Keira T.
      • Ito H.
      • Nakagawa H.
      • Nishimura S.
      Separation of isomeric 2-aminopyridine derivatized N-glycans and N-glycopeptides of human serum immunoglobulin G by using a zwitterionic type of hydrophilic-interaction chromatography.
      ). Furthermore, greater retention is observed with increasing glycan size/complexity, and chromatographic distinction between the 3-arm and 6-arm isomers of monogalactosylated species is often possible because of the slightly greater retention of the 3-arm isomer (
      • Takegawa Y.
      • Deguchi K.
      • Keira T.
      • Ito H.
      • Nakagawa H.
      • Nishimura S.
      Separation of isomeric 2-aminopyridine derivatized N-glycans and N-glycopeptides of human serum immunoglobulin G by using a zwitterionic type of hydrophilic-interaction chromatography.
      ,
      • Omtvedt L.A.
      • Royle L.
      • Husby G.
      • Sletten K.
      • Radcliffe C.M.
      • Harvey D.J.
      • Dwek R.A.
      • Rudd P.M.
      Glycan analysis of monoclonal antibodies secreted in deposition disorders indicates that subsets of plasma cells differentially process IgG glycans.
      ). The high organic modifier content applied in HILIC mobile phases makes this separation technique particularly well suited for MS interfacing.
      Fast and straightforward analysis of IgG Fc glycosylation is achieved by enriching the tryptic Fc glycopeptides using HILIC solid phase extraction followed by direct-infusion ESI-MS(/MS) (
      • Neue K.
      • Mormann M.
      • Peter-Katalinic J.
      • Pohlentz G.
      Elucidation of glycoprotein structures by unspecific proteolysis and direct nanoESI mass spectrometric analysis of ZIC-HILIC-enriched glycopeptides.
      ,
      • Reusch D.
      • Haberger M.
      • Selman M.H.
      • Bulau P.
      • Deelder A.M.
      • Wuhrer M.
      • Engler N.
      High-throughput work flow for IgG Fc-glycosylation analysis of biotechnological samples.
      ). Alternatively, MALDI-MS of purified Fc-glycopeptides can be performed with either positive- or negative-mode ionization (
      • Kolarich D.
      • Jensen P.H.
      • Altmann F.
      • Packer N.H.
      Determination of site-specific glycan heterogeneity on glycoproteins.
      ,
      • Mysling S.
      • Palmisano G.
      • Hojrup P.
      • Thaysen-Andersen M.
      Utilizing ion-pairing hydrophilic interaction chromatography solid phase extraction for efficient glycopeptide enrichment in glycoproteomics.
      ,
      • Selman M.H.
      • McDonnell L.A.
      • Palmblad M.
      • Ruhaak L.R.
      • Deelder A.M.
      • Wuhrer M.
      Immunoglobulin G glycopeptide profiling by matrix-assisted laser desorption ionization Fourier transform ion cyclotron resonance mass spectrometry.
      ,
      • Wada Y.
      • Tajiri M.
      • Yoshida S.
      Hydrophilic affinity isolation and MALDI multiple-stage tandem mass spectrometry of glycopeptides for glycoproteomics.
      ). When combined with delayed-extraction TOF detection, MALDI analysis of sialylated Fc-glycopeptides might result in a vast degree of in-source decay, largely dependent on the matrix chosen for sample preparation. When α-cyano-4-hydroxycinnamic acid is used, sialylated species are almost completely degraded. In contrast, the analysis of sialylated Fc glycopeptides is possible with 2,5-dihydroxybenzoic acid and 4-chloro-α-cyanocinnamic acid, especially when combined with negative-mode ionization (
      • Selman M.H.
      • Hoffmann M.
      • Zauner G.
      • McDonnell L.A.
      • Balog C.I.
      • Rapp E.
      • Deelder A.M.
      • Wuhrer M.
      MALDI-TOF-MS analysis of sialylated glycans and glycopeptides using 4-chloro-alpha-cyanocinnamic acid matrix.
      ). Interestingly, MALDI Fourier transform ion cyclotron resonance (FTICR) MS, which features an intermediate-pressure ion source, allows the registration of sialylated glycopeptides with both 2,5-dihydroxybenzoic acid and α-cyano-4-hydroxycinnamic acid (
      • Selman M.H.
      • McDonnell L.A.
      • Palmblad M.
      • Ruhaak L.R.
      • Deelder A.M.
      • Wuhrer M.
      Immunoglobulin G glycopeptide profiling by matrix-assisted laser desorption ionization Fourier transform ion cyclotron resonance mass spectrometry.
      ). This may be attributed to the efficient cooling of nascent ions, which limits in-source decay (
      • O'Connor P.B.
      • Budnik B.A.
      • Ivleva V.B.
      • Kaur P.
      • Moyer S.C.
      • Pittman J.L.
      • Costello C.E.
      A high pressure matrix-assisted laser desorption ion source for Fourier transform mass spectrometry designed to accommodate large targets with diverse surfaces.
      ). Although direct infusion-ESI-MS and MALDI-MS have superior throughput relative to LC/MS approaches, the accurate relative quantification of polyclonal human IgG Fc glycoforms might be compromised by the presence of isomeric tryptic glycopeptides of different IgG subclasses. However, this is not an issue with mAbs. These high-throughput approaches show particularly high potential for biopharmaceutical quality control and fermentation monitoring (
      • Reusch D.
      • Haberger M.
      • Selman M.H.
      • Bulau P.
      • Deelder A.M.
      • Wuhrer M.
      • Engler N.
      High-throughput work flow for IgG Fc-glycosylation analysis of biotechnological samples.
      ). It has to be taken into account, however, that unlike human polyclonal IgG, which is over 99% glycosylated in the Fc moiety, biotechnologically produced IgG might contain Fc peptides holding the consensus N-glycosylation sequence but lacking glycosylation. As most non-glycosylated peptides will be lost upon HILIC solid phase extraction, RP and porous graphitized-carbon-based sample preparations might be advantageous for such samples in order to allow the simultaneous analysis of glycosylated and non-glycosylated versions of the Fc peptide covering the N-glycosylation site.
      Another strategy for analyzing IgG Fc glycosylation on biopharmaceuticals involves ESI-high-resolution-MS(/MS) of intact mAbs or Fc portions prepared via reduction or enzymatic digestion (
      • Reusch D.
      • Haberger M.
      • Selman M.H.
      • Bulau P.
      • Deelder A.M.
      • Wuhrer M.
      • Engler N.
      High-throughput work flow for IgG Fc-glycosylation analysis of biotechnological samples.
      ,
      • Bondarenko P.V.
      • Second T.P.
      • Zabrouskov V.
      • Makarov A.A.
      • Zhang Z.
      Mass measurement and top-down HPLC/MS analysis of intact monoclonal antibodies on a hybrid linear quadrupole ion trap-Orbitrap mass spectrometer.
      ,
      • Fornelli L.
      • Damoc E.
      • Thomas P.M.
      • Kelleher N.L.
      • Aizikov K.
      • Denisov E.
      • Makarov A.
      • Tsybin Y.O.
      Analysis of intact monoclonal antibody IgG1 by electron transfer dissociation orbitrap FTMS.
      ). The high mass accuracy obtained with current high-resolution mass spectrometers allows one to determine the glycoform composition on intact monoclonal antibodies based on accurate mass with a typical 15-ppm (
      • Bondarenko P.V.
      • Second T.P.
      • Zabrouskov V.
      • Makarov A.A.
      • Zhang Z.
      Mass measurement and top-down HPLC/MS analysis of intact monoclonal antibodies on a hybrid linear quadrupole ion trap-Orbitrap mass spectrometer.
      ) to 2-ppm (
      • Fornelli L.
      • Damoc E.
      • Thomas P.M.
      • Kelleher N.L.
      • Aizikov K.
      • Denisov E.
      • Makarov A.
      • Tsybin Y.O.
      Analysis of intact monoclonal antibody IgG1 by electron transfer dissociation orbitrap FTMS.
      ) mass accuracy error. Moreover, up to 33% peptide sequence coverage has been reported for an intact commercial recombinant IgG using an LC-ESI-electron transfer dissociation high-resolution MS/MS approach in which time-domain transients recorded in different LC-MS/MS experiments were averaged prior to Fourier transform signal processing (
      • Fornelli L.
      • Damoc E.
      • Thomas P.M.
      • Kelleher N.L.
      • Aizikov K.
      • Denisov E.
      • Makarov A.
      • Tsybin Y.O.
      Analysis of intact monoclonal antibody IgG1 by electron transfer dissociation orbitrap FTMS.
      ). Although intact glycoprotein analysis works well to profile Fc glycoforms on mAbs, it might not be applicable for highly complex samples such as human polyclonal IgG.
      To elucidate the role of IgG Fc glycosylation in autoimmunity, inflammatory diseases, and cancer, many studies use murine disease models. Fc glycosylation of murine IgGs, however, considerably differs from that of human IgGs with regard to sialylation, fucosylation, and bisection (Fig. 2). The sialic acid on murine IgG appears to be exclusively N-glycolylneuraminic acid, whereas human IgG exclusively exhibits N-acetylneuraminic acid (
      • Raju T.S.
      Terminal sugars of Fc glycans influence antibody effector functions of IgGs.
      ,
      • Blomme B.
      • Van S.C.
      • Grassi P.
      • Haslam S.M.
      • Dell A.
      • Callewaert N.
      • Van V.H.
      Alterations of serum protein N-glycosylation in two mouse models of chronic liver disease are hepatocyte and not B cell driven.
      ). Moreover, serum-derived murine IgG1 and IgG2a/b both show high levels of disialylated Fc glycopeptides (signal at m/z 1127.41; Fig. 2A) (
      • Blomme B.
      • Van S.C.
      • Grassi P.
      • Haslam S.M.
      • Dell A.
      • Callewaert N.
      • Van V.H.
      Alterations of serum protein N-glycosylation in two mouse models of chronic liver disease are hepatocyte and not B cell driven.
      ,
      • Mizuochi T.
      • Hamako J.
      • Titani K.
      Structures of the sugar chains of mouse immunoglobulin G.
      ,
      • Mizuochi T.
      • Hamako J.
      • Nose M.
      • Titani K.
      Structural changes in the oligosaccharide chains of IgG in autoimmune MRL/Mp-lpr/lpr mice.
      ). On human IgG, disialylated Fc N-glycopeptides have only recently been reported for recombinantly expressed mAb at a low relative abundance (
      • Reusch D.
      • Haberger M.
      • Selman M.H.
      • Bulau P.
      • Deelder A.M.
      • Wuhrer M.
      • Engler N.
      High-throughput work flow for IgG Fc-glycosylation analysis of biotechnological samples.
      ), but they can also be found on polyclonal IgG from human circulation, albeit at a low relative intensity (signal at m/z 1180.79; Fig. 2B). Fucosylation on murine IgG is even higher than on human IgG, with non-fucosylated glycoforms being almost completely missing. Also, bisected species are lacking on murine IgG Fc portions, making the overall glycoform repertoire of murine IgG much more restricted than that of human IgG. Thus, IgG Fc glycosylation variation observed in murine models might not directly translate to the human situation.
      IgG samples that are biotechnologically produced or derived from human circulation are generally available in relatively large amounts (often microgram quantities), and the sensitivity of MS methods is therefore not an issue. It has been demonstrated, however, that IgG subpopulations might diverge considerably from total serum IgG in terms of Fc glycosylation profiles (
      • Scherer H.U.
      • Wang J.
      • Toes R.E.
      • van der Woude D.
      • Koeleman C.A.
      • de Boer A.R.
      • Huizinga T.W.
      • Deelder A.M.
      • Wuhrer M.
      Immunoglobulin 1 (IgG1) Fc-glycosylation profiling of anti-citrullinated peptide antibodies from human serum.
      ,
      • Scherer H.U.
      • van der Woude D.
      • Ioan-Facsinay A.
      • el Bannoudi H.
      • Trouw L.A.
      • Wang J.
      • Haupl T.
      • Burmester G.R.
      • Deelder A.M.
      • Huizinga T.W.
      • Wuhrer M.
      • Toes R.E.
      Glycan profiling of anti-citrullinated protein antibodies isolated from human serum and synovial fluid.
      ,
      • Selman M.H.
      • de Jong S.E.
      • Soonawala D.
      • Kroon F.P.
      • Adegnika A.A.
      • Deelder A.M.
      • Hokke C.H.
      • Yazdanbakhsh M.
      • Wuhrer M.
      Changes in antigen-specific IgG1 Fc N-glycosylation upon influenza and tetanus vaccination.
      ,
      • Wuhrer M.
      • Porcelijn L.
      • Kapur R.
      • Koeleman C.A.
      • Deelder A.
      • de Haas M.
      • Vidarsson G.
      Regulated glycosylation patterns of IgG during alloimmune responses against human platelet antigens.
      ). Thus, the analysis of specific subpopulations of IgG has been found to be rewarding and has repeatedly revealed skewed glycosylation profiles that might have a profound influence on the biological activity of, for example, pathogenic autoantibodies and alloantibodies (
      • Scherer H.U.
      • Wang J.
      • Toes R.E.
      • van der Woude D.
      • Koeleman C.A.
      • de Boer A.R.
      • Huizinga T.W.
      • Deelder A.M.
      • Wuhrer M.
      Immunoglobulin 1 (IgG1) Fc-glycosylation profiling of anti-citrullinated peptide antibodies from human serum.
      ,
      • Scherer H.U.
      • van der Woude D.
      • Ioan-Facsinay A.
      • el Bannoudi H.
      • Trouw L.A.
      • Wang J.
      • Haupl T.
      • Burmester G.R.
      • Deelder A.M.
      • Huizinga T.W.
      • Wuhrer M.
      • Toes R.E.
      Glycan profiling of anti-citrullinated protein antibodies isolated from human serum and synovial fluid.
      ,
      • Wuhrer M.
      • Porcelijn L.
      • Kapur R.
      • Koeleman C.A.
      • Deelder A.
      • de Haas M.
      • Vidarsson G.
      Regulated glycosylation patterns of IgG during alloimmune responses against human platelet antigens.
      ). Notably, these antibodies often may be obtained in only minute amounts by means of affinity purification, and conventional nano-LC/MS has in some cases been found to have insufficient sensitivity to analyze their Fc glycosylation. A recently reported transient-isotachophoresis separation in neutrally coated capillaries with a porous sheathless sprayer interfaced with an ultra-high-resolution TOF mass spectrometer addressed this issue, bringing the lower limit of detection down to ∼20 amol (
      • Heemskerk A.A.
      • Wuhrer M.
      • Busnel J.M.
      • Koeleman C.A.
      • Selman M.H.
      • Vidarsson G.
      • Kapur R.
      • Schoenmaker B.
      • Derks R.J.
      • Deelder A.M.
      • Mayboroda O.A.
      Coupling porous sheathless interface mass spectrometry with transient-isotachophoresis in neutral capillaries for improved sensitivity in glycopeptide analysis.
      ). This high sensitivity was reached as a result of reduced ion suppression, which is typical of ESI at very low flow rates such as those used with capillary electrophoresis sheathless ESI-MS (
      • Busnel J.M.
      • Schoenmaker B.
      • Ramautar R.
      • Carrasco-Pancorbo A.
      • Ratnayake C.
      • Feitelson J.S.
      • Chapman J.D.
      • Deelder A.M.
      • Mayboroda O.A.
      High capacity capillary electrophoresis-electrospray ionization mass spectrometry: coupling a porous sheathless interface with transient-isotachophoresis.
      ).

      Fab Glycosylation Analysis

      Besides the conserved N-glycosylation sites on the Fc portion, additional carbohydrate chains can be linked to the hypervariable regions of Ig. For instance, between 15% and 25% of IgG molecules isolated from the serum of healthy human subjects have been reported to carry N-glycans on their variable domains (
      • Anumula K.R.
      Quantitative glycan profiling of normal human plasma derived immunoglobulin and its fragments Fab and Fc.
      ,
      • Holland M.
      • Yagi H.
      • Takahashi N.
      • Kato K.
      • Savage C.O.
      • Goodall D.M.
      • Jefferis R.
      Differential glycosylation of polyclonal IgG, IgG-Fc and IgG-Fab isolated from the sera of patients with ANCA-associated systemic vasculitis.
      ,
      • Stadlmann J.
      • Pabst M.
      • Altmann F.
      Analytical and functional aspects of antibody sialylation.
      ). IgG populations with Fab glycans have been called asymmetric antibodies and were found to be bound by the lectin concanavalin A (
      • Malan B.I.
      • Gentile T.
      • Angelucci J.
      • Pividori J.
      • Guala M.C.
      • Binaghi R.A.
      • Margni R.A.
      IgG asymmetric molecules with antipaternal activity isolated from sera and placenta of pregnant human.
      ,
      • Margni R.A.
      • Malan B.I.
      Paradoxical behavior of asymmetric IgG antibodies.
      ). Interestingly, the amount of asymmetric IgG was found to increase during pregnancy, as well as after the treatment of antibody-producing cells with hormones (e.g. progesterone, estrogen) and cytokines (e.g. IL-6) (
      • Canellada A.
      • Blois S.
      • Gentile T.
      • Margni Idehu R.A.
      In vitro modulation of protective antibody responses by estrogen, progesterone and interleukin-6.
      ,
      • Gutierrez G.
      • Malan B.I.
      • Margni R.A.
      The placental regulatory factor involved in the asymmetric IgG antibody synthesis responds to IL-6 features.
      ,
      • Zenclussen A.C.
      • Gentile T.
      • Kortebani G.
      • Mazzolli A.
      • Margni R.
      Asymmetric antibodies and pregnancy.
      ). More recently, HPLC and MS analyses of Fab-linked glycans from human serum IgG have revealed primarily complex-type biantennary N-glycans with high contents of core-fucose (∼80%), bisecting GlcNAc (>50%), and sialic acid (∼80%) (
      • Anumula K.R.
      Quantitative glycan profiling of normal human plasma derived immunoglobulin and its fragments Fab and Fc.
      ,
      • Holland M.
      • Yagi H.
      • Takahashi N.
      • Kato K.
      • Savage C.O.
      • Goodall D.M.
      • Jefferis R.
      Differential glycosylation of polyclonal IgG, IgG-Fc and IgG-Fab isolated from the sera of patients with ANCA-associated systemic vasculitis.
      ,
      • Stadlmann J.
      • Weber A.
      • Pabst M.
      • Anderle H.
      • Kunert R.
      • Ehrlich H.J.
      • Peter S.H.
      • Altmann F.
      A close look at human IgG sialylation and subclass distribution after lectin fractionation.
      ). Depending on their structures and locations, the Fab glycans may influence IgG effector functions by increasing or decreasing the affinity for the antigen (
      • Arnold J.N.
      • Wormald M.R.
      • Sim R.B.
      • Rudd P.M.
      • Dwek R.A.
      The impact of glycosylation on the biological function and structure of human immunoglobulins.
      ). One report furthermore suggests that Fab glycosylation could modulate antibody half-life (
      • Huang L.
      • Biolsi S.
      • Bales K.R.
      • Kuchibhotla U.
      Impact of variable domain glycosylation on antibody clearance: an LC/MS characterization.
      ). Therefore, a better understanding of IgG functionality requires a detailed analysis of Fab specific glycosylation.
      The choice of an appropriate strategy for the analysis of IgG Fab glycosylation is determined by the biological source (monoclonal IgG versus polyclonal IgG antibodies). Monoclonal IgGs, which exhibit well-defined Fab glycosylation sites, can be analyzed at the level of glycopeptides and IgG portions (Fc, Fab, heavy and light chains), as well as after the selective release of Fab-glycans using glycosidases. LC/MS allows the analysis of both Fc- and Fab-glycopeptides at the same time, thereby revealing site-specific N-glycan microheterogeneity on therapeutic antibodies (
      • Toyama A.
      • Nakagawa H.
      • Matsuda K.
      • Sato T.A.
      • Nakamura Y.
      • Ueda K.
      Quantitative structural characterization of local N-glycan microheterogeneity in therapeutic antibodies by energy-resolved oxonium ion monitoring.
      ). Alternatively, the glycosylation of heavy and light chains of IgG mAbs can be studied via LC/MS or direct-infusion ESI-MS after reduction (
      • Chevreux G.
      • Tilly N.
      • Bihoreau N.
      Fast analysis of recombinant monoclonal antibodies using IdeS proteolytic digestion and electrospray mass spectrometry.
      ,
      • Mimura Y.
      • Ashton P.R.
      • Takahashi N.
      • Harvey D.J.
      • Jefferis R.
      Contrasting glycosylation profiles between Fab and Fc of a human IgG protein studied by electrospray ionization mass spectrometry.
      ). The separation of Fab and Fc fragments of IgG is generally accomplished using the enzymes papain (
      • Holland M.
      • Yagi H.
      • Takahashi N.
      • Kato K.
      • Savage C.O.
      • Goodall D.M.
      • Jefferis R.
      Differential glycosylation of polyclonal IgG, IgG-Fc and IgG-Fab isolated from the sera of patients with ANCA-associated systemic vasculitis.
      ,
      • Huang L.
      • Biolsi S.
      • Bales K.R.
      • Kuchibhotla U.
      Impact of variable domain glycosylation on antibody clearance: an LC/MS characterization.
      ,
      • Mimura Y.
      • Ashton P.R.
      • Takahashi N.
      • Harvey D.J.
      • Jefferis R.
      Contrasting glycosylation profiles between Fab and Fc of a human IgG protein studied by electrospray ionization mass spectrometry.
      ,
      • Qian J.
      • Liu T.
      • Yang L.
      • Daus A.
      • Crowley R.
      • Zhou Q.
      Structural characterization of N-linked oligosaccharides on monoclonal antibody cetuximab by the combination of orthogonal matrix-assisted laser desorption/ionization hybrid quadrupole-quadrupole time-of-flight tandem mass spectrometry and sequential enzymatic digestion.
      ) or pepsin (
      • Holland M.
      • Yagi H.
      • Takahashi N.
      • Kato K.
      • Savage C.O.
      • Goodall D.M.
      • Jefferis R.
      Differential glycosylation of polyclonal IgG, IgG-Fc and IgG-Fab isolated from the sera of patients with ANCA-associated systemic vasculitis.
      ). Papain cleaves IgG just above the disulfide bridges between the two heavy chains, resulting in two Fab portions and an Fc portion of similar molecular weight (∼50 kDa each). Pepsin cleaves below the disulfide bridges, generating a F(ab′)2 (±100 kDa) and two ½ Fc portions (±25 kDa each). In 2002, a streptococcal cysteine proteinase, IdeS, was reported to cleave IgG specifically at a unique site below the hinge region, leading to the formation of F(ab′)2 fragments with great yield and specificity (
      • von Pawel-Rammingen U.
      • Johansson B.P.
      • Bjorck L.
      IdeS, a novel streptococcal cysteine proteinase with unique specificity for immunoglobulin G.
      ). A recombinant version of this enzyme is now commercially available under the brand name FabRICATOR (Genovis, Lund, Sweden). A multitude of approaches have been used to purify F(ab) and F(ab′)2 fragments. After pepsin digestion, Fc glycopeptides and F(ab′)2 portions were separated using size exclusion (
      • Holland M.
      • Yagi H.
      • Takahashi N.
      • Kato K.
      • Savage C.O.
      • Goodall D.M.
      • Jefferis R.
      Differential glycosylation of polyclonal IgG, IgG-Fc and IgG-Fab isolated from the sera of patients with ANCA-associated systemic vasculitis.
      ). Papain digestion was followed by ion exchange chromatography (
      • Holland M.
      • Yagi H.
      • Takahashi N.
      • Kato K.
      • Savage C.O.
      • Goodall D.M.
      • Jefferis R.
      Differential glycosylation of polyclonal IgG, IgG-Fc and IgG-Fab isolated from the sera of patients with ANCA-associated systemic vasculitis.
      ) or affinity chromatography using Protein A (
      • Stadlmann J.
      • Weber A.
      • Pabst M.
      • Anderle H.
      • Kunert R.
      • Ehrlich H.J.
      • Peter S.H.
      • Altmann F.
      A close look at human IgG sialylation and subclass distribution after lectin fractionation.
      ,
      • Qian J.
      • Liu T.
      • Yang L.
      • Daus A.
      • Crowley R.
      • Zhou Q.
      Structural characterization of N-linked oligosaccharides on monoclonal antibody cetuximab by the combination of orthogonal matrix-assisted laser desorption/ionization hybrid quadrupole-quadrupole time-of-flight tandem mass spectrometry and sequential enzymatic digestion.
      ). In all cases, the Fab-linked N-glycans were released using PNGase F and analyzed via HPLC or MS.
      Another way to separately analyze Fc and Fab glycans of a mAb is to release them from the entire IgG molecule using discriminating glycosidases and/or enzymatic conditions. For example, PNGase F and endoglycosidase F2 were reported to selectively release, in native condition, the Fc and Fab glycans, respectively (
      • Mimura Y.
      • Ashton P.R.
      • Takahashi N.
      • Harvey D.J.
      • Jefferis R.
      Contrasting glycosylation profiles between Fab and Fc of a human IgG protein studied by electrospray ionization mass spectrometry.
      ).
      Polyclonal IgGs exhibit a vast diversity of amino acid sequences of the variable regions created during somatic hypermutation, resulting in a multitude of Fab-glycosylation sites differing in number and location, as well as in the nature of their glycans. This enormous heterogeneity complicates, if not precludes, Fab glycosylation analysis at the glycopeptide level. Consequently, Fab glycosylation analysis of polyclonal IgG has hitherto relied on the analysis of released glycans from parts of IgG or from entire IgG molecules.
      Recently, a method using sequential enzymatic release of Fc glycans and Fab glycans has been reported (
      • Anumula K.R.
      Quantitative glycan profiling of normal human plasma derived immunoglobulin and its fragments Fab and Fc.
      ). Fab glycans, but not Fc sugars, were found to be resistant to PNGase F cleavage under native conditions (
      • Anumula K.R.
      Quantitative glycan profiling of normal human plasma derived immunoglobulin and its fragments Fab and Fc.
      ). Therefore, IgG Fc glycans were first released under native conditions, and after IgG isolation, denaturing conditions allowed the liberation of Fab glycans.
      For all techniques that use released glycans, a major drawback is that samples have to be extremely pure. Fc glycosylation is close to 100%, whereas Fab glycosylation is found on a only minor portion of polyclonal IgGs. Minor Fc contamination in Fab samples can bias the results. Fab and Fc glycosylation analysis at the released glycan level might be similarly compromised by the presence of other glycoprotein contaminants. This underlines the importance of highly specific purification methods.

      Immunoglobulin A Glycosylation Analysis

      Immunoglobulin A has several N- (IgA1 and 2) and O-glycosylation sites (IgA1 only; see Fig. 1), and both N- and O-glycosylation have been analyzed at the released glycan level (
      • Mattu T.S.
      • Pleass R.J.
      • Willis A.C.
      • Kilian M.
      • Wormald M.R.
      • Lellouch A.C.
      • Rudd P.M.
      • Woof J.M.
      • Dwek R.A.
      The glycosylation and structure of human serum IgA1, Fab, and Fc regions and the role of N-glycosylation on Fc alpha receptor interactions.
      ,
      • Royle L.
      • Roos A.
      • Harvey D.J.
      • Wormald M.R.
      • van Gijlswijk-Janssen D.
      • Redwan e.
      • Wilson I.A.
      • Daha M.R.
      • Dwek R.A.
      • Rudd P.M.
      Secretory IgA N- and O-glycans provide a link between the innate and adaptive immune systems.
      ). In secretory fluids, such as mucosa and milk, two IgA molecules are dimerized by the N-glycosylated secretory component and the joining (J-)chain (
      • Deshpande N.
      • Jensen P.H.
      • Packer N.H.
      • Kolarich D.
      GlycoSpectrumScan: fishing glycopeptides from MS spectra of protease digests of human colostrum sIgA.
      ).
      Site-specific N-glycosylation analysis of IgA has been done at the glycopeptide level after employing Asp-N endoproteinase (
      • Tanaka A.
      • Iwase H.
      • Hiki Y.
      • Kokubo T.
      • Ishii-Karakasa I.
      • Toma K.
      • Kobayashi Y.
      • Hotta K.
      Evidence for a site-specific fucosylation of N-linked oligosaccharide of immunoglobulin A1 from normal human serum.
      ). Two N-glycopeptides were identified, and the peptide sequences were obtained by means of Edman degradation. Based on the calculated masses of these sequences, different glycan compositions were deduced from MALDI-TOF-MS of desialylated glycopeptides. Differential treatment with galactosidase and fucosidase, as well as two-dimensional HPLC on released glycans using C18 and amide columns, revealed fully galactosylated complex-type biantennary structures with or without bisecting GlcNAc and fucose (
      • Tanaka A.
      • Iwase H.
      • Hiki Y.
      • Kokubo T.
      • Ishii-Karakasa I.
      • Toma K.
      • Kobayashi Y.
      • Hotta K.
      Evidence for a site-specific fucosylation of N-linked oligosaccharide of immunoglobulin A1 from normal human serum.
      ). More recently, tryptic glycopeptides of size-exclusion-chromatography-purified IgA1 have been analyzed using LC-FTICR-MS, with sequence confirmation using electron capture dissociation (ECD)-FTICR-MS/MS (
      • Gomes M.M.
      • Wall S.B.
      • Takahashi K.
      • Novak J.
      • Renfrow M.B.
      • Herr A.B.
      Analysis of IgA1 N-glycosylation and its contribution to FcalphaRI binding.
      ). Glycan compositions and linkages were established via gas-liquid chromatography. Interestingly, bi-, tri- and tetra-antennary complex type glycans were observed (
      • Gomes M.M.
      • Wall S.B.
      • Takahashi K.
      • Novak J.
      • Renfrow M.B.
      • Herr A.B.
      Analysis of IgA1 N-glycosylation and its contribution to FcalphaRI binding.
      ). N-glycosylation analysis of secretory IgA from human colostrum has recently been performed at the glycopeptide level using in-gel trypsin digestion and subsequent LC/MS and LC-MS/MS (
      • Deshpande N.
      • Jensen P.H.
      • Packer N.H.
      • Kolarich D.
      GlycoSpectrumScan: fishing glycopeptides from MS spectra of protease digests of human colostrum sIgA.
      ), revealing pronounced site-specific differences in glycosylation.
      Also, the O-glycosylation of IgA has been extensively studied at the glycopeptide level (
      • Deshpande N.
      • Jensen P.H.
      • Packer N.H.
      • Kolarich D.
      GlycoSpectrumScan: fishing glycopeptides from MS spectra of protease digests of human colostrum sIgA.
      ,
      • Renfrow M.B.
      • Cooper H.J.
      • Tomana M.
      • Kulhavy R.
      • Hiki Y.
      • Toma K.
      • Emmett M.R.
      • Mestecky J.
      • Marshall A.G.
      • Novak J.
      Determination of aberrant O-glycosylation in the IgA1 hinge region by electron capture dissociation fourier transform-ion cyclotron resonance mass spectrometry.
      ,
      • Renfrow M.B.
      • Mackay C.L.
      • Chalmers M.J.
      • Julian B.A.
      • Mestecky J.
      • Kilian M.
      • Poulsen K.
      • Emmett M.R.
      • Marshall A.G.
      • Novak J.
      Analysis of O-glycan heterogeneity in IgA1 myeloma proteins by Fourier transform ion cyclotron resonance mass spectrometry: implications for IgA nephropathy.
      ,
      • Takahashi K.
      • Wall S.B.
      • Suzuki H.
      • Smith A.D.
      • Hall S.
      • Poulsen K.
      • Kilian M.
      • Mobley J.A.
      • Julian B.A.
      • Mestecky J.
      • Novak J.
      • Renfrow M.B.
      Clustered O-glycans of IgA1: defining macro- and microheterogeneity by use of electron capture/transfer dissociation.
      ,
      • Wada Y.
      • Dell A.
      • Haslam S.M.
      • Tissot B.
      • Canis K.
      • Azadi P.
      • Backstrom M.
      • Costello C.E.
      • Hansson G.C.
      • Hiki Y.
      • Ishihara M.
      • Ito H.
      • Kakehi K.
      • Karlsson N.
      • Hayes C.E.
      • Kato K.
      • Kawasaki N.
      • Khoo K.H.
      • Kobayashi K.
      • Kolarich D.
      • Kondo A.
      • Lebrilla C.
      • Nakano M.
      • Narimatsu H.
      • Novak J.
      • Novotny M.V.
      • Ohno E.
      • Packer N.H.
      • Palaima E.
      • Renfrow M.B.
      • Tajiri M.
      • Thomsson K.A.
      • Yagi H.
      • Yu S.Y.
      • Taniguchi N.
      Comparison of methods for profiling O-glycosylation: Human Proteome Organisation Human Disease Glycomics/Proteome Initiative multi-institutional study of IgA1.
      ,
      • Wada Y.
      • Tajiri M.
      • Ohshima S.
      Quantitation of saccharide compositions of O-glycans by mass spectrometry of glycopeptides and its application to rheumatoid arthritis.
      ). Specific O-glycosylation changes were found in IgA nephropathy (
      • Novak J.
      • Julian B.A.
      • Tomana M.
      • Mestecky J.
      IgA glycosylation and IgA immune complexes in the pathogenesis of IgA nephropathy.
      ). More specifically, aberrantly glycosylated IgA1, with Gal-deficient hinge region (HR) O-glycans, plays a pivotal role in the pathogenesis of IgA nephropathy (
      • Renfrow M.B.
      • Mackay C.L.
      • Chalmers M.J.
      • Julian B.A.
      • Mestecky J.
      • Kilian M.
      • Poulsen K.
      • Emmett M.R.
      • Marshall A.G.
      • Novak J.
      Analysis of O-glycan heterogeneity in IgA1 myeloma proteins by Fourier transform ion cyclotron resonance mass spectrometry: implications for IgA nephropathy.
      ,
      • Takahashi K.
      • Wall S.B.
      • Suzuki H.
      • Smith A.D.
      • Hall S.
      • Poulsen K.
      • Kilian M.
      • Mobley J.A.
      • Julian B.A.
      • Mestecky J.
      • Novak J.
      • Renfrow M.B.
      Clustered O-glycans of IgA1: defining macro- and microheterogeneity by use of electron capture/transfer dissociation.
      ,
      • Novak J.
      • Julian B.A.
      • Mestecky J.
      • Renfrow M.B.
      Glycosylation of IgA1 and pathogenesis of IgA nephropathy.
      ). Renfrow and coworkers showed IgA1 O-glycan heterogeneity via the use of FTICR-MS and LC-FTICR-MS to obtain accurate mass profiles of IgA1 HR glycopeptides from three different IgA1 myeloma proteins (
      • Renfrow M.B.
      • Mackay C.L.
      • Chalmers M.J.
      • Julian B.A.
      • Mestecky J.
      • Kilian M.
      • Poulsen K.
      • Emmett M.R.
      • Marshall A.G.
      • Novak J.
      Analysis of O-glycan heterogeneity in IgA1 myeloma proteins by Fourier transform ion cyclotron resonance mass spectrometry: implications for IgA nephropathy.
      ). Additionally, in that study, the first ECD fragmentation approach on an individual IgA1 O-glycopeptide from an IgA1 HR preparation that was reproducible for each IgA1 myeloma protein was obtained (Fig. 3). These results suggest that future analyses of IgA1 HRs from IgA nephropathy patients and healthy controls should be feasible.
      Figure thumbnail gr3
      Fig. 3ECD MS/MS spectrum of an HR IgA peptide. The O-glycosylated tryptic HR peptide ion [peptide + 2 GalNAc + 2 Gal + 4H]4+ from IgA1 was analyzed via ESI-FTICR ECD MS/MS. Sites of O-glycosylation were identified from series of differentially glycosylated product ions. The product ions localize one GalNAc-Gal disaccharide unambiguously to T225. A second GalNAc-Gal is narrowed to three possible sites, T228, S230, or S232. The remaining eight Ser/Thr residues are eliminated as sites of O-glycan attachment (in blue). N-terminal fragment ions (c and b) and C-terminal fragment ions (z) are indicated above and below the IgA1 HR sequence, respectively. Not all detected ECD fragments are labeled in the spectrum. Squares, GalNAc; circles, Gal. Taken from Renfrow et al. with permission (
      • Renfrow M.B.
      • Mackay C.L.
      • Chalmers M.J.
      • Julian B.A.
      • Mestecky J.
      • Kilian M.
      • Poulsen K.
      • Emmett M.R.
      • Marshall A.G.
      • Novak J.
      Analysis of O-glycan heterogeneity in IgA1 myeloma proteins by Fourier transform ion cyclotron resonance mass spectrometry: implications for IgA nephropathy.
      ).
      Novel strategies for the analysis of clustered O-glycans involve the use of a combination of IgA-specific proteases and trypsin and ECD-FTICR-MS/MS. They provide a variety of IgA1 HR fragments that allow the unambiguous localization of all O-glycosylation sites for the six most abundant glycoforms, leading to the identification of Gal-deficient sites (
      • Takahashi K.
      • Wall S.B.
      • Suzuki H.
      • Smith A.D.
      • Hall S.
      • Poulsen K.
      • Kilian M.
      • Mobley J.A.
      • Julian B.A.
      • Mestecky J.
      • Novak J.
      • Renfrow M.B.
      Clustered O-glycans of IgA1: defining macro- and microheterogeneity by use of electron capture/transfer dissociation.
      ). Additionally, the published protocol was adapted for on-line LC-ECD-MS/MS and LC–electron transfer dissociation–MS/MS analysis. This work appears to be a relevant clinical approach for defining the molecular events leading to the pathogenesis of a chronic kidney disease, and at the same time it might be generally applicable for the analysis of clustered sites of O-glycosylation (
      • Takahashi K.
      • Wall S.B.
      • Suzuki H.
      • Smith A.D.
      • Hall S.
      • Poulsen K.
      • Kilian M.
      • Mobley J.A.
      • Julian B.A.
      • Mestecky J.
      • Novak J.
      • Renfrow M.B.
      Clustered O-glycans of IgA1: defining macro- and microheterogeneity by use of electron capture/transfer dissociation.
      ).

      PERSPECTIVES

      As demonstrated extensively for IgG, as well as for some IgA, a detailed structural analysis of N- and O-glycosylation is required in order for one to understand their three-dimensional structures and immune functions. To our knowledge, the glycosylation of other Igs (IgD, IgE, IgM) has hitherto not been addressed at the glycoproteomic level. The numerous O-glycosylation sites in the IgD HR and N-glycosylation sites (≥5 N-glycosylation sites) in IgM and IgE make their comprehensive glycosylation analysis at the glycopeptide level challenging. Additionally, the analysis of IgE and IgD from human circulation is particularly demanding, as these antibodies are generally present only in minute amounts (
      • Arnold J.N.
      • Radcliffe C.M.
      • Wormald M.R.
      • Royle L.
      • Harvey D.J.
      • Crispin M.
      • Dwek R.A.
      • Sim R.B.
      • Rudd P.M.
      The glycosylation of human serum IgD and IgE and the accessibility of identified oligomannose structures for interaction with mannan-binding lectin.
      ). For IgE, glycoproteomic analysis would be needed in order to allocate its complex type and oligomannosidic glycans to their specific site(s) and analyze the NxS site on position 383, which has been predicted to be unoccupied (
      • Dorrington K.J.
      • Bennich H.H.
      Structure-function relationships in human immunoglobulin E.
      ). A particular analytical challenge will be the analysis of the variable region glycosylation of Ig subclasses other than IgG.
      Ig glycosylation studies are routinely done in many different labs, and thus the amount of data produced is increasing tremendously. A recent approach combining genome-wide association and high-throughput glycomics analysis of plasma samples from 2705 individuals in three population cohorts showed that common variants in certain genes can influence N-glycan levels in human plasma (
      • Lauc G.
      • Essafi A.
      • Huffman J.E.
      • Hayward C.
      • Knezevic A.
      • Kattla J.J.
      • Polasek O.
      • Gornik O.
      • Vitart V.
      • Abrahams J.L.
      • Pucic M.
      • Novokmet M.
      • Redzic I.
      • Campbell S.
      • Wild S.H.
      • Borovecki F.
      • Wang W.
      • Kolcic I.
      • Zgaga L.
      • Gyllensten U.
      • Wilson J.F.
      • Wright A.F.
      • Hastie N.D.
      • Campbell H.
      • Rudd P.M.
      • Rudan I.
      Genomics meets glycomics-the first GWAS study of human N-Glycome identifies HNF1alpha as a master regulator of plasma protein fucosylation.
      ). Based on a follow-up study, a high-throughput isolation and glycosylation analysis of IgG variability and heritability of the IgG glycome in three different populations was published (
      • Pucic M.
      • Knezevic A.
      • Vidic J.
      • Adamczyk B.
      • Novokmet M.
      • Polasek O.
      • Gornik O.
      • Supraha-Goreta S.
      • Wormald M.R.
      • Redzic I.
      • Campbell H.
      • Wright A.
      • Hastie N.D.
      • Wilson J.F.
      • Rudan I.
      • Wuhrer M.
      • Rudd P.M.
      • Josic D.
      • Lauc G.
      High throughput isolation and glycosylation analysis of IgG-variability and heritability of the IgG glycome in three isolated human populations.
      ). Although a variety of associations of clinical and physiological parameters with Ig glycosylation have been established, we believe that many more processes and diseases are marked by Ig glycosylation changes and that we have seen only the tip of the iceberg. Future Ig glycosylation profiling at the site-specific level by means of the mass spectrometric analysis of glycopeptides, when applied to human disease cohorts as well as in vitro and in vivo models of immunological processes, is expected to provide valuable new insights into the modulatory role of Ig glycosylation.

      REFERENCES

        • Arnold J.N.
        • Wormald M.R.
        • Sim R.B.
        • Rudd P.M.
        • Dwek R.A.
        The impact of glycosylation on the biological function and structure of human immunoglobulins.
        Annu. Rev. Immunol. 2007; 25: 21-50
        • Borrok M.J.
        • Jung S.T.
        • Kang T.H.
        • Monzingo A.F.
        • Georgiou G.
        Revisiting the role of glycosylation in the structure of human IgG fc.
        ACS Chem. Biol. 2012; 7: 1596-1602
        • Krapp S.
        • Mimura Y.
        • Jefferis R.
        • Huber R.
        • Sondermann P.
        Structural analysis of human IgG-Fc glycoforms reveals a correlation between glycosylation and structural integrity.
        J. Mol. Biol. 2003; 325: 979-989
        • Jung S.T.
        • Reddy S.T.
        • Kang T.H.
        • Borrok M.J.
        • Sandlie I.
        • Tucker P.W.
        • Georgiou G.
        Aglycosylated IgG variants expressed in bacteria that selectively bind FcgammaRI potentiate tumor cell killing by monocyte-dendritic cells.
        Proc. Natl. Acad. Sci. U.S.A. 2010; 107: 604-609
        • Nesspor T.C.
        • Raju T.S.
        • Chin C.N.
        • Vafa O.
        • Brezski R.J.
        Avidity confers FcgammaR binding and immune effector function to aglycosylated immunoglobulin G1.
        J. Mol. Recognit. 2012; 25: 147-154
        • Tao M.H.
        • Morrison S.L.
        Studies of aglycosylated chimeric mouse-human IgG. Role of carbohydrate in the structure and effector functions mediated by the human IgG constant region.
        J. Immunol. 1989; 143: 2595-2601
        • Parekh R.B.
        • Dwek R.A.
        • Sutton B.J.
        • Fernandes D.L.
        • Leung A.
        • Stanworth D.
        • Rademacher T.W.
        • Mizuochi T.
        • Taniguchi T.
        • Matsuta K.
        • Takeuchi F.
        • Nagano Y.
        • Miyamoto T.
        • Kobata A.
        Association of rheumatoid arthritis and primary osteoarthritis with changes in the glycosylation pattern of total serum IgG.
        Nature. 1985; 316: 452-457
        • Parekh R.
        • Isenberg D.
        • Rook G.
        • Roitt I.
        • Dwek R.
        • Rademacher T.
        A comparative analysis of disease-associated changes in the galactosylation of serum IgG.
        J. Autoimmun. 1989; 2: 101-114
        • Ercan A.
        • Barnes M.G.
        • Hazen M.
        • Tory H.
        • Henderson L.
        • Dedeoglu F.
        • Fuhlbrigge R.C.
        • Grom A.
        • Holm I.A.
        • Kellogg M.
        • Kim S.
        • Adamczyk B.
        • Rudd P.M.
        • Son M.B.
        • Sundel R.P.
        • Foell D.
        • Glass D.N.
        • Thompson S.D.
        • Nigrovic P.A.
        Multiple juvenile idiopathic arthritis subtypes demonstrate proinflammatory IgG glycosylation.
        Arthritis Rheum. 2012; 64: 3025-3033
        • Pucic M.
        • Knezevic A.
        • Vidic J.
        • Adamczyk B.
        • Novokmet M.
        • Polasek O.
        • Gornik O.
        • Supraha-Goreta S.
        • Wormald M.R.
        • Redzic I.
        • Campbell H.
        • Wright A.
        • Hastie N.D.
        • Wilson J.F.
        • Rudan I.
        • Wuhrer M.
        • Rudd P.M.
        • Josic D.
        • Lauc G.
        High throughput isolation and glycosylation analysis of IgG-variability and heritability of the IgG glycome in three isolated human populations.
        Mol. Cell. Proteomics. 2011; 10M111.010090
        • Ruhaak L.R.
        • Uh H.W.
        • Beekman M.
        • Koeleman C.A.
        • Hokke C.H.
        • Westendorp R.G.
        • Wuhrer M.
        • Houwing-Duistermaat J.J.
        • Slagboom P.E.
        • Deelder A.M.
        Decreased levels of bisecting GlcNAc glycoforms of IgG are associated with human longevity.
        PLoS. One. 2010; 5: e12566
        • Saldova R.
        • Royle L.
        • Radcliffe C.M.
        • Abd Hamid U.M.
        • Evans R.
        • Arnold J.N.
        • Banks R.E.
        • Hutson R.
        • Harvey D.J.
        • Antrobus R.
        • Petrescu S.M.
        • Dwek R.A.
        • Rudd P.M.
        Ovarian cancer is associated with changes in glycosylation in both acute-phase proteins and IgG.
        Glycobiology. 2007; 17: 1344-1356
        • van de Geijn F.E.
        • Wuhrer M.
        • Selman M.H.
        • Willemsen S.P.
        • de Man Y.A.
        • Deelder A.M.
        • Hazes J.M.
        • Dolhain R.J.
        Immunoglobulin G galactosylation and sialylation are associated with pregnancy-induced improvement of rheumatoid arthritis and the postpartum flare: results from a large prospective cohort study.
        Arthritis Res. Ther. 2009; 11: R193
        • Bones J.
        • Mittermayr S.
        • O'Donoghue N.
        • Guttman A.
        • Rudd P.M.
        Ultra performance liquid chromatographic profiling of serum N-glycans for fast and efficient identification of cancer associated alterations in glycosylation.
        Anal. Chem. 2010; 82: 10208-10215
        • Huhn C.
        • Selman M.H.
        • Ruhaak L.R.
        • Deelder A.M.
        • Wuhrer M.
        IgG glycosylation analysis.
        Proteomics. 2009; 9: 882-913
        • Gindzienska-Sieskiewicz E.
        • Klimiuk P.A.
        • Kisiel D.G.
        • Gindzienski A.
        • Sierakowski S.
        The changes in monosaccharide composition of immunoglobulin G in the course of rheumatoid arthritis.
        Clin. Rheumatol. 2007; 26: 685-690
        • van Zeben D.
        • Rook G.A.
        • Hazes J.M.
        • Zwinderman A.H.
        • Zhang Y.
        • Ghelani S.
        • Rademacher T.W.
        • Breedveld F.C.
        Early agalactosylation of IgG is associated with a more progressive disease course in patients with rheumatoid arthritis: results of a follow-up study.
        Br. J. Rheumatol. 1994; 33: 36-43
        • Jefferis R.
        • Lund J.
        • Pound J.D.
        IgG-Fc-mediated effector functions: molecular definition of interaction sites for effector ligands and the role of glycosylation.
        Immunol. Rev. 1998; 163: 59-76
        • Kaneko Y.
        • Nimmerjahn F.
        • Ravetch J.V.
        Anti-inflammatory activity of immunoglobulin G resulting from Fc sialylation.
        Science. 2006; 313: 670-673
        • Nimmerjahn F.
        • Anthony R.M.
        • Ravetch J.V.
        Agalactosylated IgG antibodies depend on cellular Fc receptors for in vivo activity.
        Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 8433-8437
        • Shields R.L.
        • Lai J.
        • Keck R.
        • O'Connell L.Y.
        • Hong K.
        • Meng Y.G.
        • Weikert S.H.
        • Presta L.G.
        Lack of fucose on human IgG1 N-linked oligosaccharide improves binding to human Fcgamma RIII and antibody-dependent cellular toxicity.
        J. Biol. Chem. 2002; 277: 26733-26740
        • Ferrara C.
        • Grau S.
        • Jager C.
        • Sondermann P.
        • Brunker P.
        • Waldhauer I.
        • Hennig M.
        • Ruf A.
        • Rufer A.C.
        • Stihle M.
        • Umana P.
        • Benz J.
        Unique carbohydrate-carbohydrate interactions are required for high affinity binding between FcgammaRIII and antibodies lacking core fucose.
        Proc. Natl. Acad. Sci. U.S.A. 2011; 108: 12669-12674
        • Zou G.
        • Ochiai H.
        • Huang W.
        • Yang Q.
        • Li C.
        • Wang L.X.
        Chemoenzymatic synthesis and Fcgamma receptor binding of homogeneous glycoforms of antibody Fc domain. Presence of a bisecting sugar moiety enhances the affinity of Fc to FcgammaIIIa receptor.
        J. Am. Chem. Soc. 2011; 133: 18975-18991
        • Salles G.
        • Morschhauser F.
        • Lamy T.
        • Milpied N.
        • Thieblemont C.
        • Tilly H.
        • Bieska G.
        • Asikanius E.
        • Carlile D.
        • Birkett J.
        • Pisa P.
        • Cartron G.
        Phase 1 study results of the type II glycoengineered humanized anti-CD20 monoclonal antibody obinutuzumab (GA101) in B-cell lymphoma patients.
        Blood. 2012; 119: 5126-5132
        • Sehn L.H.
        • Assouline S.E.
        • Stewart D.A.
        • Mangel J.
        • Gascoyne R.D.
        • Fine G.
        • Frances-Lasserre S.
        • Carlile D.J.
        • Crump M.
        A phase 1 study of obinutuzumab induction followed by 2 years of maintenance in patients with relapsed CD20-positive B-cell malignancies.
        Blood. 2012; 119: 5118-5125
        • Malhotra R.
        • Wormald M.R.
        • Rudd P.M.
        • Fischer P.B.
        • Dwek R.A.
        • Sim R.B.
        Glycosylation changes of IgG associated with rheumatoid arthritis can activate complement via the mannose-binding protein.
        Nat. Med. 1995; 1: 237-243
        • Raju T.S.
        Terminal sugars of Fc glycans influence antibody effector functions of IgGs.
        Curr. Opin. Immunol. 2008; 20: 471-478
        • Karsten C.M.
        • Pandey M.K.
        • Figge J.
        • Kilchenstein R.
        • Taylor P.R.
        • Rosas M.
        • McDonald J.U.
        • Orr S.J.
        • Berger M.
        • Petzold D.
        • Blanchard V.
        • Winkler A.
        • Hess C.
        • Reid D.M.
        • Majoul I.V.
        • Strait R.T.
        • Harris N.L.
        • Kohl G.
        • Wex E.
        • Ludwig R.
        • Zillikens D.
        • Nimmerjahn F.
        • Finkelman F.D.
        • Brown G.D.
        • Ehlers M.
        • Kohl J.
        Anti-inflammatory activity of IgG1 mediated by Fc galactosylation and association of FcgammaRIIB and dectin-1.
        Nat. Med. 2012; 18: 1401-1406
        • Anthony R.M.
        • Ravetch J.V.
        A novel role for the IgG Fc glycan: the anti-inflammatory activity of sialylated IgG Fcs.
        J. Clin. Immunol. 2010; 30: S9-S14
        • Anthony R.M.
        • Kobayashi T.
        • Wermeling F.
        • Ravetch J.V.
        Intravenous gammaglobulin suppresses inflammation through a novel T(H)2 pathway.
        Nature. 2011; 475: 110-113
        • Chen G.
        • Wang Y.
        • Qiu L.
        • Qin X.
        • Liu H.
        • Wang X.
        • Wang Y.
        • Song G.
        • Li F.
        • Guo Y.
        • Li F.
        • Guo S.
        • Li Z.
        Human IgG Fc-glycosylation profiling reveals associations with age, sex, female sex hormones and thyroid cancer.
        J. Proteomics. 2012; 75: 2824-2834
        • Prados M.B.
        • La B.J.
        • Szekeres-Bartho J.
        • Caramelo J.
        • Miranda S.
        Progesterone induces a switch in oligosaccharyltransferase isoform expression: consequences on IgG N-glycosylation.
        Immunol. Lett. 2011; 137: 28-37
        • Wang J.
        • Balog C.I.
        • Stavenhagen K.
        • Koeleman C.A.
        • Scherer H.U.
        • Selman M.H.
        • Deelder A.M.
        • Huizinga T.W.
        • Toes R.E.
        • Wuhrer M.
        Fc-glycosylation of IgG1 is modulated by B-cell stimuli.
        Mol. Cell. Proteomics. 2011; 10M110.004655
        • Novak J.
        • Julian B.A.
        • Tomana M.
        • Mestecky J.
        IgA glycosylation and IgA immune complexes in the pathogenesis of IgA nephropathy.
        Semin. Nephrol. 2008; 28: 78-87
        • Dalpathado D.S.
        • Desaire H.
        Glycopeptide analysis by mass spectrometry.
        Analyst. 2008; 133: 731-738
        • Morelle W.
        • Michalski J.C.
        Analysis of protein glycosylation by mass spectrometry.
        Nat. Protoc. 2007; 2: 1585-1602
        • Kolarich D.
        • Jensen P.H.
        • Altmann F.
        • Packer N.H.
        Determination of site-specific glycan heterogeneity on glycoproteins.
        Nat. Protoc. 2012; 7: 1285-1298
        • Zauner G.
        • Deelder A.M.
        • Wuhrer M.
        Recent advances in hydrophilic interaction liquid chromatography (HILIC) for structural glycomics.
        Electrophoresis. 2011; 32: 3456-3466
        • Nwosu C.C.
        • Seipert R.R.
        • Strum J.S.
        • Hua S.S.
        • An H.J.
        • Zivkovic A.M.
        • German B.J.
        • Lebrilla C.B.
        Simultaneous and extensive site-specific N- and O-glycosylation analysis in protein mixtures.
        J. Proteome Res. 2011; 10: 2612-2624
        • Mechref Y.
        • Hu Y.
        • Garcia A.
        • Zhou S.
        • Desantos-Garcia J.L.
        • Hussein A.
        Defining putative glycan cancer biomarkers by MS.
        Bioanalysis. 2012; 4: 2457-2469
        • Mechref Y.
        • Muzikar J.
        • Novotny M.V.
        Comprehensive assessment of N-glycans derived from a murine monoclonal antibody: a case for multimethodological approach.
        Electrophoresis. 2005; 26: 2034-2046
        • Ruhaak L.R.
        • Zauner G.
        • Huhn C.
        • Bruggink C.
        • Deelder A.M.
        • Wuhrer M.
        Glycan labeling strategies and their use in identification and quantification.
        Anal. Bioanal. Chem. 2010; 397: 3457-3481
        • Harvey D.J.
        Analysis of carbohydrates and glycoconjugates by matrix-assisted laser desorption/ionization mass spectrometry: an update for 2007–2008.
        Mass Spectrom. Rev. 2012; 31: 183-311
        • Leymarie N.
        • Zaia J.
        Effective use of mass spectrometry for glycan and glycopeptide structural analysis.
        Anal. Chem. 2012; 84: 3040-3048
        • Dard P.
        • Lefranc M.P.
        • Osipova L.
        • Sanchez-Mazas A.
        DNA sequence variability of IGHG3 alleles associated to the main G3m haplotypes in human populations.
        Eur. J. Hum. Genet. 2001; 9: 765-772
        • Jefferis R.
        • Lefranc M.P.
        Human immunoglobulin allotypes: possible implications for immunogenicity.
        MAbs. 2009; 1: 332-338
        • Balbin M.
        • Grubb A.
        • de Lange G.G.
        • Grubb R.
        DNA sequences specific for Caucasian G3m(b) and(g) allotypes: allotyping at the genomic level.
        Immunogenetics. 1994; 39: 187-193
        • Perdivara I.
        • Deterding L.J.
        • Cozma C.
        • Tomer K.B.
        • Przybylski M.
        Glycosylation profiles of epitope-specific anti-beta-amyloid antibodies revealed by liquid chromatography-mass spectrometry.
        Glycobiology. 2009; 19: 958-970
        • Stadlmann J.
        • Pabst M.
        • Kolarich D.
        • Kunert R.
        • Altmann F.
        Analysis of immunoglobulin glycosylation by LC-ESI-MS of glycopeptides and oligosaccharides.
        Proteomics. 2008; 8: 2858-2871
        • Wuhrer M.
        • Stam J.C.
        • van de Geijn F.E.
        • Koeleman C.A.
        • Verrips C.T.
        • Dolhain R.J.
        • Hokke C.H.
        • Deelder A.M.
        Glycosylation profiling of immunoglobulin G (IgG) subclasses from human serum.
        Proteomics. 2007; 7: 4070-4081
        • Selman M.H.
        • Derks R.J.
        • Bondt A.
        • Palmblad M.
        • Schoenmaker B.
        • Koeleman C.A.
        • van de Geijn F.E.
        • Dolhain R.J.
        • Deelder A.M.
        • Wuhrer M.
        Fc specific IgG glycosylation profiling by robust nano-reverse phase HPLC-MS using a sheath-flow ESI sprayer interface.
        J. Proteomics. 2012; 75: 1318-1329
        • Gilar M.
        • Yu Y.Q.
        • Ahn J.
        • Xie H.
        • Han H.
        • Ying W.
        • Qian X.
        Characterization of glycoprotein digests with hydrophilic interaction chromatography and mass spectrometry.
        Anal. Biochem. 2011; 417: 80-88
        • Singh C.
        • Zampronio C.G.
        • Creese A.J.
        • Cooper H.J.
        Higher energy collision dissociation (HCD) product ion-triggered electron transfer dissociation (ETD) mass spectrometry for the analysis of N-linked glycoproteins.
        J. Proteome Res. 2012; 11: 4517-4525
        • Takegawa Y.
        • Deguchi K.
        • Keira T.
        • Ito H.
        • Nakagawa H.
        • Nishimura S.
        Separation of isomeric 2-aminopyridine derivatized N-glycans and N-glycopeptides of human serum immunoglobulin G by using a zwitterionic type of hydrophilic-interaction chromatography.
        J. Chromatogr. A. 2006; 1113: 177-181
        • Omtvedt L.A.
        • Royle L.
        • Husby G.
        • Sletten K.
        • Radcliffe C.M.
        • Harvey D.J.
        • Dwek R.A.
        • Rudd P.M.
        Glycan analysis of monoclonal antibodies secreted in deposition disorders indicates that subsets of plasma cells differentially process IgG glycans.
        Arthritis Rheum. 2006; 54: 3433-3440
        • Neue K.
        • Mormann M.
        • Peter-Katalinic J.
        • Pohlentz G.
        Elucidation of glycoprotein structures by unspecific proteolysis and direct nanoESI mass spectrometric analysis of ZIC-HILIC-enriched glycopeptides.
        J. Proteome Res. 2011; 10: 2248-2260
        • Reusch D.
        • Haberger M.
        • Selman M.H.
        • Bulau P.
        • Deelder A.M.
        • Wuhrer M.
        • Engler N.
        High-throughput work flow for IgG Fc-glycosylation analysis of biotechnological samples.
        Anal. Biochem. 2012; 432: 82-89
        • Mysling S.
        • Palmisano G.
        • Hojrup P.
        • Thaysen-Andersen M.
        Utilizing ion-pairing hydrophilic interaction chromatography solid phase extraction for efficient glycopeptide enrichment in glycoproteomics.
        Anal. Chem. 2010; 82: 5598-5609
        • Selman M.H.
        • McDonnell L.A.
        • Palmblad M.
        • Ruhaak L.R.
        • Deelder A.M.
        • Wuhrer M.
        Immunoglobulin G glycopeptide profiling by matrix-assisted laser desorption ionization Fourier transform ion cyclotron resonance mass spectrometry.
        Anal. Chem. 2010; 82: 1073-1081
        • Wada Y.
        • Tajiri M.
        • Yoshida S.
        Hydrophilic affinity isolation and MALDI multiple-stage tandem mass spectrometry of glycopeptides for glycoproteomics.
        Anal. Chem. 2004; 76: 6560-6565
        • Selman M.H.
        • Hoffmann M.
        • Zauner G.
        • McDonnell L.A.
        • Balog C.I.
        • Rapp E.
        • Deelder A.M.
        • Wuhrer M.
        MALDI-TOF-MS analysis of sialylated glycans and glycopeptides using 4-chloro-alpha-cyanocinnamic acid matrix.
        Proteomics. 2012; 12: 1337-1348
        • O'Connor P.B.
        • Budnik B.A.
        • Ivleva V.B.
        • Kaur P.
        • Moyer S.C.
        • Pittman J.L.
        • Costello C.E.
        A high pressure matrix-assisted laser desorption ion source for Fourier transform mass spectrometry designed to accommodate large targets with diverse surfaces.
        J. Am. Soc. Mass Spectrom. 2004; 15: 128-132
        • Bondarenko P.V.
        • Second T.P.
        • Zabrouskov V.
        • Makarov A.A.
        • Zhang Z.
        Mass measurement and top-down HPLC/MS analysis of intact monoclonal antibodies on a hybrid linear quadrupole ion trap-Orbitrap mass spectrometer.
        J. Am. Soc. Mass Spectrom. 2009; 20: 1415-1424
        • Fornelli L.
        • Damoc E.
        • Thomas P.M.
        • Kelleher N.L.
        • Aizikov K.
        • Denisov E.
        • Makarov A.
        • Tsybin Y.O.
        Analysis of intact monoclonal antibody IgG1 by electron transfer dissociation orbitrap FTMS.
        Mol. Cell. Proteomics. 2012; 11: 1758-1767
        • Blomme B.
        • Van S.C.
        • Grassi P.
        • Haslam S.M.
        • Dell A.
        • Callewaert N.
        • Van V.H.
        Alterations of serum protein N-glycosylation in two mouse models of chronic liver disease are hepatocyte and not B cell driven.
        Am. J. Physiol. Gastrointest. Liver Physiol. 2011; 300: G833-G842
        • Mizuochi T.
        • Hamako J.
        • Titani K.
        Structures of the sugar chains of mouse immunoglobulin G.
        Arch. Biochem. Biophys. 1987; 257: 387-394
        • Mizuochi T.
        • Hamako J.
        • Nose M.
        • Titani K.
        Structural changes in the oligosaccharide chains of IgG in autoimmune MRL/Mp-lpr/lpr mice.
        J. Immunol. 1990; 145: 1794-1798
        • Scherer H.U.
        • Wang J.
        • Toes R.E.
        • van der Woude D.
        • Koeleman C.A.
        • de Boer A.R.
        • Huizinga T.W.
        • Deelder A.M.
        • Wuhrer M.
        Immunoglobulin 1 (IgG1) Fc-glycosylation profiling of anti-citrullinated peptide antibodies from human serum.
        Proteomics Clin. Appl. 2009; 3: 106-115
        • Scherer H.U.
        • van der Woude D.
        • Ioan-Facsinay A.
        • el Bannoudi H.
        • Trouw L.A.
        • Wang J.
        • Haupl T.
        • Burmester G.R.
        • Deelder A.M.
        • Huizinga T.W.
        • Wuhrer M.
        • Toes R.E.
        Glycan profiling of anti-citrullinated protein antibodies isolated from human serum and synovial fluid.
        Arthritis Rheum. 2010; 62: 1620-1629
        • Selman M.H.
        • de Jong S.E.
        • Soonawala D.
        • Kroon F.P.
        • Adegnika A.A.
        • Deelder A.M.
        • Hokke C.H.
        • Yazdanbakhsh M.
        • Wuhrer M.
        Changes in antigen-specific IgG1 Fc N-glycosylation upon influenza and tetanus vaccination.
        Mol. Cell. Proteomics. 2012; 11M111.014563
        • Wuhrer M.
        • Porcelijn L.
        • Kapur R.
        • Koeleman C.A.
        • Deelder A.
        • de Haas M.
        • Vidarsson G.
        Regulated glycosylation patterns of IgG during alloimmune responses against human platelet antigens.
        J. Proteome Res. 2009; 8: 450-456
        • Heemskerk A.A.
        • Wuhrer M.
        • Busnel J.M.
        • Koeleman C.A.
        • Selman M.H.
        • Vidarsson G.
        • Kapur R.
        • Schoenmaker B.
        • Derks R.J.
        • Deelder A.M.
        • Mayboroda O.A.
        Coupling porous sheathless interface mass spectrometry with transient-isotachophoresis in neutral capillaries for improved sensitivity in glycopeptide analysis.
        Electrophoresis. 2013; 34: 383-387
        • Busnel J.M.
        • Schoenmaker B.
        • Ramautar R.
        • Carrasco-Pancorbo A.
        • Ratnayake C.
        • Feitelson J.S.
        • Chapman J.D.
        • Deelder A.M.
        • Mayboroda O.A.
        High capacity capillary electrophoresis-electrospray ionization mass spectrometry: coupling a porous sheathless interface with transient-isotachophoresis.
        Anal. Chem. 2010; 82: 9476-9483
        • Anumula K.R.
        Quantitative glycan profiling of normal human plasma derived immunoglobulin and its fragments Fab and Fc.
        J. Immunol. Methods. 2012; 382: 167-176
        • Holland M.
        • Yagi H.
        • Takahashi N.
        • Kato K.
        • Savage C.O.
        • Goodall D.M.
        • Jefferis R.
        Differential glycosylation of polyclonal IgG, IgG-Fc and IgG-Fab isolated from the sera of patients with ANCA-associated systemic vasculitis.
        Biochim. Biophys. Acta. 2006; 1760: 669-677
        • Stadlmann J.
        • Pabst M.
        • Altmann F.
        Analytical and functional aspects of antibody sialylation.
        J. Clin. Immunol. 2010; 30: 15-19
        • Malan B.I.
        • Gentile T.
        • Angelucci J.
        • Pividori J.
        • Guala M.C.
        • Binaghi R.A.
        • Margni R.A.
        IgG asymmetric molecules with antipaternal activity isolated from sera and placenta of pregnant human.
        J. Reprod. Immunol. 1991; 20: 129-140
        • Margni R.A.
        • Malan B.I.
        Paradoxical behavior of asymmetric IgG antibodies.
        Immunol. Rev. 1998; 163: 77-87
        • Canellada A.
        • Blois S.
        • Gentile T.
        • Margni Idehu R.A.
        In vitro modulation of protective antibody responses by estrogen, progesterone and interleukin-6.
        Am. J. Reprod. Immunol. 2002; 48: 334-343
        • Gutierrez G.
        • Malan B.I.
        • Margni R.A.
        The placental regulatory factor involved in the asymmetric IgG antibody synthesis responds to IL-6 features.
        J. Reprod. Immunol. 2001; 49: 21-32
        • Zenclussen A.C.
        • Gentile T.
        • Kortebani G.
        • Mazzolli A.
        • Margni R.
        Asymmetric antibodies and pregnancy.
        Am. J. Reprod. Immunol. 2001; 45: 289-294
        • Stadlmann J.
        • Weber A.
        • Pabst M.
        • Anderle H.
        • Kunert R.
        • Ehrlich H.J.
        • Peter S.H.
        • Altmann F.
        A close look at human IgG sialylation and subclass distribution after lectin fractionation.
        Proteomics. 2009; 9: 4143-4153
        • Huang L.
        • Biolsi S.
        • Bales K.R.
        • Kuchibhotla U.
        Impact of variable domain glycosylation on antibody clearance: an LC/MS characterization.
        Anal. Biochem. 2006; 349: 197-207
        • Toyama A.
        • Nakagawa H.
        • Matsuda K.
        • Sato T.A.
        • Nakamura Y.
        • Ueda K.
        Quantitative structural characterization of local N-glycan microheterogeneity in therapeutic antibodies by energy-resolved oxonium ion monitoring.
        Anal. Chem. 2012; 84: 9655-9662
        • Chevreux G.
        • Tilly N.
        • Bihoreau N.
        Fast analysis of recombinant monoclonal antibodies using IdeS proteolytic digestion and electrospray mass spectrometry.
        Anal. Biochem. 2011; 415: 212-214
        • Mimura Y.
        • Ashton P.R.
        • Takahashi N.
        • Harvey D.J.
        • Jefferis R.
        Contrasting glycosylation profiles between Fab and Fc of a human IgG protein studied by electrospray ionization mass spectrometry.
        J. Immunol. Methods. 2007; 326: 116-126
        • Qian J.
        • Liu T.
        • Yang L.
        • Daus A.
        • Crowley R.
        • Zhou Q.
        Structural characterization of N-linked oligosaccharides on monoclonal antibody cetuximab by the combination of orthogonal matrix-assisted laser desorption/ionization hybrid quadrupole-quadrupole time-of-flight tandem mass spectrometry and sequential enzymatic digestion.
        Anal. Biochem. 2007; 364: 8-18
        • von Pawel-Rammingen U.
        • Johansson B.P.
        • Bjorck L.
        IdeS, a novel streptococcal cysteine proteinase with unique specificity for immunoglobulin G.
        EMBO J. 2002; 21: 1607-1615
        • Mattu T.S.
        • Pleass R.J.
        • Willis A.C.
        • Kilian M.
        • Wormald M.R.
        • Lellouch A.C.
        • Rudd P.M.
        • Woof J.M.
        • Dwek R.A.
        The glycosylation and structure of human serum IgA1, Fab, and Fc regions and the role of N-glycosylation on Fc alpha receptor interactions.
        J. Biol. Chem. 1998; 273: 2260-2272
        • Royle L.
        • Roos A.
        • Harvey D.J.
        • Wormald M.R.
        • van Gijlswijk-Janssen D.
        • Redwan e.
        • Wilson I.A.
        • Daha M.R.
        • Dwek R.A.
        • Rudd P.M.
        Secretory IgA N- and O-glycans provide a link between the innate and adaptive immune systems.
        J. Biol. Chem. 2003; 278: 20140-20153
        • Deshpande N.
        • Jensen P.H.
        • Packer N.H.
        • Kolarich D.
        GlycoSpectrumScan: fishing glycopeptides from MS spectra of protease digests of human colostrum sIgA.
        J. Proteome Res. 2010; 9: 1063-1075
        • Tanaka A.
        • Iwase H.
        • Hiki Y.
        • Kokubo T.
        • Ishii-Karakasa I.
        • Toma K.
        • Kobayashi Y.
        • Hotta K.
        Evidence for a site-specific fucosylation of N-linked oligosaccharide of immunoglobulin A1 from normal human serum.
        Glycoconj. J. 1998; 15: 995-1000
        • Gomes M.M.
        • Wall S.B.
        • Takahashi K.
        • Novak J.
        • Renfrow M.B.
        • Herr A.B.
        Analysis of IgA1 N-glycosylation and its contribution to FcalphaRI binding.
        Biochemistry. 2008; 47: 11285-11299
        • Renfrow M.B.
        • Cooper H.J.
        • Tomana M.
        • Kulhavy R.
        • Hiki Y.
        • Toma K.
        • Emmett M.R.
        • Mestecky J.
        • Marshall A.G.
        • Novak J.
        Determination of aberrant O-glycosylation in the IgA1 hinge region by electron capture dissociation fourier transform-ion cyclotron resonance mass spectrometry.
        J. Biol. Chem. 2005; 280: 19136-19145
        • Renfrow M.B.
        • Mackay C.L.
        • Chalmers M.J.
        • Julian B.A.
        • Mestecky J.
        • Kilian M.
        • Poulsen K.
        • Emmett M.R.
        • Marshall A.G.
        • Novak J.
        Analysis of O-glycan heterogeneity in IgA1 myeloma proteins by Fourier transform ion cyclotron resonance mass spectrometry: implications for IgA nephropathy.
        Anal. Bioanal. Chem. 2007; 389: 1397-1407
        • Takahashi K.
        • Wall S.B.
        • Suzuki H.
        • Smith A.D.
        • Hall S.
        • Poulsen K.
        • Kilian M.
        • Mobley J.A.
        • Julian B.A.
        • Mestecky J.
        • Novak J.
        • Renfrow M.B.
        Clustered O-glycans of IgA1: defining macro- and microheterogeneity by use of electron capture/transfer dissociation.
        Mol. Cell. Proteomics. 2010; 9: 2545-2557
        • Wada Y.
        • Dell A.
        • Haslam S.M.
        • Tissot B.
        • Canis K.
        • Azadi P.
        • Backstrom M.
        • Costello C.E.
        • Hansson G.C.
        • Hiki Y.
        • Ishihara M.
        • Ito H.
        • Kakehi K.
        • Karlsson N.
        • Hayes C.E.
        • Kato K.
        • Kawasaki N.
        • Khoo K.H.
        • Kobayashi K.
        • Kolarich D.
        • Kondo A.
        • Lebrilla C.
        • Nakano M.
        • Narimatsu H.
        • Novak J.
        • Novotny M.V.
        • Ohno E.
        • Packer N.H.
        • Palaima E.
        • Renfrow M.B.
        • Tajiri M.
        • Thomsson K.A.
        • Yagi H.
        • Yu S.Y.
        • Taniguchi N.
        Comparison of methods for profiling O-glycosylation: Human Proteome Organisation Human Disease Glycomics/Proteome Initiative multi-institutional study of IgA1.
        Mol. Cell. Proteomics. 2010; 9: 719-727
        • Wada Y.
        • Tajiri M.
        • Ohshima S.
        Quantitation of saccharide compositions of O-glycans by mass spectrometry of glycopeptides and its application to rheumatoid arthritis.
        J. Proteome Res. 2010; 9: 1367-1373
        • Novak J.
        • Julian B.A.
        • Mestecky J.
        • Renfrow M.B.
        Glycosylation of IgA1 and pathogenesis of IgA nephropathy.
        Semin. Immunopathol. 2012; 34: 365-382
        • Arnold J.N.
        • Radcliffe C.M.
        • Wormald M.R.
        • Royle L.
        • Harvey D.J.
        • Crispin M.
        • Dwek R.A.
        • Sim R.B.
        • Rudd P.M.
        The glycosylation of human serum IgD and IgE and the accessibility of identified oligomannose structures for interaction with mannan-binding lectin.
        J. Immunol. 2004; 173: 6831-6840
        • Dorrington K.J.
        • Bennich H.H.
        Structure-function relationships in human immunoglobulin E.
        Immunol. Rev. 1978; 41: 3-25
        • Lauc G.
        • Essafi A.
        • Huffman J.E.
        • Hayward C.
        • Knezevic A.
        • Kattla J.J.
        • Polasek O.
        • Gornik O.
        • Vitart V.
        • Abrahams J.L.
        • Pucic M.
        • Novokmet M.
        • Redzic I.
        • Campbell S.
        • Wild S.H.
        • Borovecki F.
        • Wang W.
        • Kolcic I.
        • Zgaga L.
        • Gyllensten U.
        • Wilson J.F.
        • Wright A.F.
        • Hastie N.D.
        • Campbell H.
        • Rudd P.M.
        • Rudan I.
        Genomics meets glycomics-the first GWAS study of human N-Glycome identifies HNF1alpha as a master regulator of plasma protein fucosylation.
        PLoS. Genet. 2010; 6: e1001256