Glycoproteomic Analysis of Antibodies
2013; Elsevier BV; Volume: 12; Issue: 4 Linguagem: Inglês
10.1074/mcp.r112.026005
ISSN1535-9484
AutoresGerhild Zauner, Maurice H. J. Selman, Albert Bondt, Yoann Rombouts, Dennis Blank, André M. Deelder, Manfred Wuhrer,
Tópico(s)Protein purification and stability
ResumoAntibody 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. 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. Immunoglobulins (Igs) 1The abbreviations used are:ECDelectron capture dissociationESIelectrospray ionizationFabfragment antigen bindingFcfragment crystallizableFTICRFourier transform ion cyclotron resonanceGalgalactosesGlcNAcN-acetylglucosamineHILIChydrophilic interaction liquid chromatographyHRhinge regionIgimmunglobulinmAbmonoclonal antibodyRPreversed-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 (1Arnold 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: 21-50Crossref PubMed Scopus (865) Google Scholar). 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 (1Arnold 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: 21-50Crossref PubMed Scopus (865) Google Scholar). 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 (2Borrok 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: 1596-1602Crossref PubMed Scopus (99) Google Scholar, 3Krapp 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: 979-989Crossref PubMed Scopus (495) Google Scholar). Their removal by glycosidases or via mutation of the glycosylation sites reduces the binding of IgG to Fc-gamma receptors (FcγR) (4Jung 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: 604-609Crossref PubMed Scopus (117) Google Scholar, 5Nesspor 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: 147-154Crossref PubMed Scopus (35) Google Scholar, 6Tao 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: 2595-2601Crossref PubMed Google Scholar). 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 (7Parekh 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: 452-457Crossref PubMed Scopus (913) Google Scholar) (see Fig. 1). electron capture dissociation electrospray ionization fragment antigen binding fragment crystallizable Fourier transform ion cyclotron resonance galactoses N-acetylglucosamine hydrophilic interaction liquid chromatography hinge region immunglobulin monoclonal antibody reversed-phase. 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. 8Parekh 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: 101-114Crossref PubMed Scopus (157) Google Scholar, 9Ercan 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: 3025-3033Crossref PubMed Scopus (0) Google Scholar, 10Pucic 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; 10M111.010090Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar, 11Ruhaak 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: e12566Crossref PubMed Scopus (81) Google Scholar, 12Saldova 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: 1344-1356Crossref PubMed Scopus (297) Google Scholar, 13van 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: R193Crossref PubMed Scopus (180) Google Scholar, 14Bones 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: 10208-10215Crossref PubMed Scopus (137) Google Scholar, 15Huhn C. Selman M.H. Ruhaak L.R. Deelder A.M. Wuhrer M. IgG glycosylation analysis.Proteomics. 2009; 9: 882-913Crossref PubMed Scopus (239) Google Scholar). For instance, an increase in IgG G0F is observed in the serum of patients with rheumatoid arthritis (7Parekh 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: 452-457Crossref PubMed Scopus (913) Google Scholar) and correlates with disease progression and severity (16Gindzienska-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: 685-690Crossref PubMed Scopus (32) Google Scholar, 17van 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: 36-43Crossref PubMed Google Scholar). 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 (18Jefferis 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: 59-76Crossref PubMed Scopus (279) Google Scholar, 19Kaneko Y. Nimmerjahn F. Ravetch J.V. Anti-inflammatory activity of immunoglobulin G resulting from Fc sialylation.Science. 2006; 313: 670-673Crossref PubMed Scopus (1220) Google Scholar, 20Nimmerjahn 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: 8433-8437Crossref PubMed Scopus (177) Google Scholar). 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 (21Shields R.L. Lai J. Keck R. O'Connell L.Y. Hong K. Meng Y.G. Weikert S.H. Presta L.G. 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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 (24Salles 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: 5126-5132Crossref PubMed Scopus (151) Google Scholar, 25Sehn 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: 5118-5125Crossref PubMed Scopus (124) Google Scholar). 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 (26Malhotra 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: 237-243Crossref PubMed Google Scholar, 27Raju T.S. Terminal sugars of Fc glycans influence antibody effector functions of IgGs.Curr. Opin. Immunol. 2008; 20: 471-478Crossref PubMed Scopus (386) Google Scholar). 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 (28Karsten 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. 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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) (31Chen 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: 2824-2834Crossref PubMed Scopus (75) Google Scholar, 32Prados 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: 28-37Crossref PubMed Scopus (21) Google Scholar, 33Wang 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; 10M110.004655Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). 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 (1Arnold 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: 21-50Crossref PubMed Scopus (865) Google Scholar). 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 (34Novak J. Julian B.A. Tomana M. Mestecky J. IgA glycosylation and IgA immune complexes in the pathogenesis of IgA nephropathy.Semin. Nephrol. 2008; 28: 78-87Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). 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 (1Arnold 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: 21-50Crossref PubMed Scopus (865) Google Scholar). 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 (15Huhn C. Selman M.H. Ruhaak L.R. Deelder A.M. Wuhrer M. IgG glycosylation analysis.Proteomics. 2009; 9: 882-913Crossref PubMed Scopus (239) Google Scholar, 35Dalpathado D.S. Desaire H. Glycopeptide analysis by mass spectrometry.Analyst. 2008; 133: 731-738Crossref PubMed Scopus (119) Google Scholar, 36Morelle W. Michalski J.C. Analysis of protein glycosylation by mass spectrometry.Nat. Protoc. 2007; 2: 1585-1602Crossref PubMed Scopus (282) Google Scholar). 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 (37Kolarich D. Jensen P.H. Altmann F. Packer N.H. Determination of site-specific glycan heterogeneity on glycoproteins.Nat. Protoc. 2012; 7: 1285-1298Crossref PubMed Scopus (139) Google Scholar). 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 (35Dalpathado D.S. Desaire H. Glycopeptide analysis by mass spectrometry.Analyst. 2008; 133: 731-738Crossref PubMed Scopus (119) Google Scholar). 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 (15Huhn C. Selman M.H. Ruhaak L.R. Deelder A.M. Wuhrer M. IgG glycosylation analysis.Proteomics. 2009; 9: 882-913Crossref PubMed Scopus (239) Google Scholar, 38Zauner G. Deelder A.M. Wuhrer M. Recent advances in hydrophilic interaction liquid chromatography (HILIC) for structural glycomics.Electrophoresis. 2011; 32: 3456-3466Crossref PubMed Scopus (128) Google Scholar, 39Nwosu 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: 2612-2624Crossref PubMed Scopus (98) Google Scholar). 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 (36Morelle W. Michalski J.C. Analysis of protein glycosylation by mass spectrometry.Nat. Protoc. 2007; 2: 1585-1602Crossref PubMed Scopus (282) Google Scholar, 40Mechref Y. Hu Y. Garcia A. Zhou S. Desantos-Garcia J.L. Hussein A. Defining putative glycan cancer biomarkers by MS.Bioanalysis. 2012; 4: 2457-2469Crossref PubMed Scopus (45) Google Scholar). 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Chem. 2012; 84: 3040-3048Crossref PubMed Scopus (134) Google Scholar). 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 (7Parekh 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: 452-457Crossref PubMed Scopus (913) Google Scholar). 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 (14Bones 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: 10208-10215Crossref PubMed Scopus (137) Google Scholar, 15Huhn C. Selman M.H. Ruhaak L.R. Deelder A.M. Wuhrer M. IgG glycosylation analysis.Proteomics. 2009; 9: 882-913Crossref PubMed Scopus (239) Google Scholar). A high-throughput isolation and glycosylation analysis of IgG was published recently by Pucic et al. (10Pucic 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; 10M111.010090Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar): 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. 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 (45Dard 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: 765-772Crossref PubMed Scopus (45) Google Scholar, 46Jefferis R. Lefranc M.P. Human immunoglobulin allotypes: possible implications for immunogenicity.MAbs. 2009; 1: 332-338Crossref PubMed Google Scholar). Analysis of IgG3 from Caucasians mainly revealed allotype G3m(b*), which exhibits a phenylalanine (F) in position 296 (47Balbin 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: 187-193Crossref PubMed Google Scholar). 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 (45Dard 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: 765-772Crossref PubMed Scopus (45) Google Scholar). Thus, allotypic variations have to be taken into account when comparing subclass-specific IgG Fc-glycosylation profiles of genetically different groups. 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 (37Kolarich D. Jensen P.H. Altmann F. Packer N.H. Determination of site-specific glycan heterogeneity on glycoproteins.Nat. Protoc. 2012; 7: 1285-1298Crossref PubMed Scopus (139) Google Scholar, 48Perdivara 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: 958-970Crossref PubMed Scopus (12) Google Scholar, 49Stadlmann J. Pabst M. Kolarich D. Kunert R. Altmann F. Analysis of immunoglobulin glycosylation by LC-ESI-MS of glycopeptides and oligosaccharides.Proteomics. 2008; 8: 2858-2871Crossref PubMed Scopus (243) Google Scholar, 50Wuhrer 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: 4070-4081Crossref PubMed Scopus (194) Google Scholar). 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 (48Perdivara 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: 958-970Crossref PubMed Scopus (12) Google Scholar, 50Wuhrer 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: 4070-4081Crossref PubMed Scopus (194) Google Scholar). We recently optimized an RP-nano-LC-ESI-M
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