Hinge-Region O-Glycosylation of Human Immunoglobulin G3 (IgG3)
2015; Elsevier BV; Volume: 14; Issue: 5 Linguagem: Inglês
10.1074/mcp.m114.047381
ISSN1535-9484
AutoresRosina Plomp, Gillian Dekkers, Yoann Rombouts, Remco Visser, Carolien A. M. Koeleman, Guinevere S. M. Lageveen‐Kammeijer, Bas C. Jansen, Theo Rispens, Paul J. Hensbergen, Gestur Vidarsson, Manfred Wuhrer,
Tópico(s)Galectins and Cancer Biology
ResumoImmunoglobulin G (IgG) is one of the most abundant proteins present in human serum and a fundamental component of the immune system. IgG3 represents ∼8% of the total amount of IgG in human serum and stands out from the other IgG subclasses because of its elongated hinge region and enhanced effector functions. This study reports partial O-glycosylation of the IgG3 hinge region, observed with nanoLC-ESI-IT-MS(/MS) analysis after proteolytic digestion. The repeat regions within the IgG3 hinge were found to be in part O-glycosylated at the threonine in the triple repeat motif. Non-, mono- and disialylated core 1-type O-glycans were detected in various IgG3 samples, both poly- and monoclonal. NanoLC-ESI-IT-MS/MS with electron transfer dissociation fragmentation and CE-MS/MS with CID fragmentation were used to determine the site of IgG3 O-glycosylation. The O-glycosylation site was further confirmed by the recombinant production of mutant IgG3 in which potential O-glycosylation sites had been knocked out.For IgG3 samples from six donors we found similar O-glycan structures and site occupancies, whereas for the same samples the conserved N-glycosylation of the Fc CH2 domain showed considerable interindividual variation. The occupancy of each of the three O-glycosylation sites was found to be ∼10% in six serum-derived IgG3 samples and ∼13% in two monoclonal IgG3 allotypes. Immunoglobulin G (IgG) is one of the most abundant proteins present in human serum and a fundamental component of the immune system. IgG3 represents ∼8% of the total amount of IgG in human serum and stands out from the other IgG subclasses because of its elongated hinge region and enhanced effector functions. This study reports partial O-glycosylation of the IgG3 hinge region, observed with nanoLC-ESI-IT-MS(/MS) analysis after proteolytic digestion. The repeat regions within the IgG3 hinge were found to be in part O-glycosylated at the threonine in the triple repeat motif. Non-, mono- and disialylated core 1-type O-glycans were detected in various IgG3 samples, both poly- and monoclonal. NanoLC-ESI-IT-MS/MS with electron transfer dissociation fragmentation and CE-MS/MS with CID fragmentation were used to determine the site of IgG3 O-glycosylation. The O-glycosylation site was further confirmed by the recombinant production of mutant IgG3 in which potential O-glycosylation sites had been knocked out. For IgG3 samples from six donors we found similar O-glycan structures and site occupancies, whereas for the same samples the conserved N-glycosylation of the Fc CH2 domain showed considerable interindividual variation. The occupancy of each of the three O-glycosylation sites was found to be ∼10% in six serum-derived IgG3 samples and ∼13% in two monoclonal IgG3 allotypes. Immunoglobulin G (IgG) is one of the most abundant proteins present in human serum and represents approximately three-quarters of the total serum immunoglobulin content (1Stoop J.W. Zegers B.J. Sander P.C. Ballieux R.E. Serum immunoglobulin levels in healthy children and adults.Clin. Experiment. Immunol. 1969; 4: 101-112PubMed Google Scholar). As the main mediator of humoral immunity and an important link between the adaptive and innate immune system, IgG is a fundamental component of the immune system. IgG consists of two heavy and light chains, linked by disulfide bonds. The protein can be subdivided into the antigen-binding (Fab) and the receptor-binding (Fc) region. There are four subclasses of IgG, all of which share an overall structure homology but differ slightly in their amino acid sequence; the quantity of the subclasses in human serum is as follows: IgG1 > 2 > 3 > 4 (2Morell A. Terry W.D. Waldmann T.A. Metabolic properties of IgG subclasses in man.J. Clin. Invest. 1970; 49: 673-680Crossref PubMed Scopus (377) Google Scholar). IgG3 represents ∼8% of the total amount of IgG in human serum (2Morell A. Terry W.D. Waldmann T.A. Metabolic properties of IgG subclasses in man.J. Clin. Invest. 1970; 49: 673-680Crossref PubMed Scopus (377) Google Scholar), and stands out from the other IgG subclasses for a number of reasons. First of all, IgG3 contains an elongated hinge region with up to a triple repeat sequence (the actual number ranging from one to three depending on the allotype (3Vidarsson G. Dekkers G. Rispens T. IgG subclasses and allotypes: From structure to effector functions.Frontiers Immunol. 2014; 5: 520Crossref PubMed Scopus (1342) Google Scholar)), which is responsible for the increased flexibility between the Fab and the Fc part, as well as the wider and more flexible angle between the two Fab arms (4Roux K.H. Strelets L. Michaelsen T.E. Flexibility of human IgG subclasses.J. 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Finally, IgG3 generally has a shorter half-life compared with the other IgG subclasses (1 versus 3 weeks) (2Morell A. Terry W.D. Waldmann T.A. Metabolic properties of IgG subclasses in man.J. Clin. Invest. 1970; 49: 673-680Crossref PubMed Scopus (377) Google Scholar). This difference was traced back to an H435R mutation that confers a positive charge at physiological pH, resulting in a decreased binding to the neonatal Fc receptor (FcRn), which is involved in recycling IgG targeted for lysosomal degradation (13Stapleton N.M. Andersen J.T. Stemerding A.M. Bjarnarson S.P. Verheul R.C. Gerritsen J. Zhao Y. Kleijer M. Sandlie I. de Haas M. Jonsdottir I. van der Schoot C.E. Vidarsson G. Competition for FcRn-mediated transport gives rise to short half-life of human IgG3 and offers therapeutic potential.Nature Commun. 2011; 2: 599Crossref PubMed Scopus (174) Google Scholar). The low-efficiency FcRn-mediated transport also gives rise to decreased levels of IgG3 in mucosal tissue and impaired transport of IgG3 across the placenta (14Einarsdottir H. Ji Y. Visser R. Mo C. Luo G. Scherjon S. van der Schoot C.E. Vidarsson G. H435-containing immunoglobulin G3 allotypes are transported efficiently across the human placenta: Implications for alloantibody-mediated diseases of the newborn.Transfusion. 2014; 54: 665-671Crossref PubMed Scopus (34) Google Scholar). These properties do not hold true for all types of IgG3 since a large number of IgG3 allotypes have been described, some of which lack the H435R substitution and have a half-life and placental transport rates similar to IgG1 (13Stapleton N.M. Andersen J.T. Stemerding A.M. Bjarnarson S.P. Verheul R.C. Gerritsen J. Zhao Y. Kleijer M. Sandlie I. de Haas M. Jonsdottir I. van der Schoot C.E. Vidarsson G. Competition for FcRn-mediated transport gives rise to short half-life of human IgG3 and offers therapeutic potential.Nature Commun. 2011; 2: 599Crossref PubMed Scopus (174) Google Scholar, 14Einarsdottir H. Ji Y. Visser R. Mo C. Luo G. Scherjon S. van der Schoot C.E. Vidarsson G. H435-containing immunoglobulin G3 allotypes are transported efficiently across the human placenta: Implications for alloantibody-mediated diseases of the newborn.Transfusion. 2014; 54: 665-671Crossref PubMed Scopus (34) Google Scholar, 15Dard 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. Human Genetics. 2001; 9: 765-772Crossref PubMed Scopus (50) Google Scholar, 16Jefferis R. Lefranc M.P. Human immunoglobulin allotypes: Possible implications for immunogenicity.mAbs. 2009; 1: 332-338Crossref PubMed Scopus (165) Google Scholar). IgG3 is more polymorphic than the other IgG subclasses, as evidenced by the high number of known allotypes (16Jefferis R. Lefranc M.P. Human immunoglobulin allotypes: Possible implications for immunogenicity.mAbs. 2009; 1: 332-338Crossref PubMed Scopus (165) Google Scholar). Most of the polymorphisms reside in the CH2 or CH3 domain, but the length of the hinge region can also display a high degree of variation. Depending on the number of sequence repeats, the hinge region can vary from 27 to 83 amino acid residues between different IgG3 allotypes (3Vidarsson G. Dekkers G. Rispens T. IgG subclasses and allotypes: From structure to effector functions.Frontiers Immunol. 2014; 5: 520Crossref PubMed Scopus (1342) Google Scholar, 16Jefferis R. Lefranc M.P. Human immunoglobulin allotypes: Possible implications for immunogenicity.mAbs. 2009; 1: 332-338Crossref PubMed Scopus (165) Google Scholar, 17Dard P. Huck S. Frippiat J.P. Lefranc G. Langaney A. Lefranc M.P. Sanchez-Mazas A. 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The role of differential IgG glycosylation in the interaction of antibodies with FcγRs in vivo.Curr. Op. Organ Transplant. 2011; 16: 7-14Crossref PubMed Scopus (69) Google Scholar, 21Kaneko Y. Nimmerjahn F. Ravetch J.V. Anti-inflammatory activity of immunoglobulin G resulting from Fc sialylation.Science. 2006; 313: 670-673Crossref PubMed Scopus (1379) Google Scholar, 22Karsten 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. Köhl G. Wex E. Ludwig R. Zillikens D. Nimmerjahn F. Finkelman F.D. Brown G.D. Ehlers M. Köhl J. Anti-inflammatory activity of IgG1 mediated by Fc galactosylation and association of Fc[gamma[RIIB and dectin-1.Nature Med. 2012; 18: 1401-1406Crossref PubMed Scopus (315) Google Scholar). O-linked glycosylation has been reported for various immunoglobulins. O-glycans are present on the hinge region of human IgA1 and IgD and mouse IgG2b (23Kim H. Yamaguchi Y. Masuda K. Matsunaga C. Yamamoto K. Irimura T. Takahashi N. Kato K. Arata Y. O-glycosylation in hinge region of mouse immunoglobulin G2b.J. Biolog. Chem. 1994; 269: 12345-12350Abstract Full Text PDF PubMed Google Scholar, 24Mattu 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 Fcα receptor interactions.J. Biolog. Chem. 1998; 273: 2260-2272Abstract Full Text Full Text PDF PubMed Scopus (353) Google Scholar, 25Takahashi N. Tetaert D. Debuire B. Lin L.C. Putnam F.W. Complete amino acid sequence of the delta heavy chain of human immunoglobulin D.Proc. Natl. Acad. Sci. U.S.A. 1982; 79: 2850-2854Crossref PubMed Scopus (41) Google Scholar). 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Commun. 1982; 105: 1066-1071Crossref PubMed Scopus (20) Google Scholar). The O-glycosylation in the hinge of murine IgG2b was observed to protect against proteolytic digestion (23Kim H. Yamaguchi Y. Masuda K. Matsunaga C. Yamamoto K. Irimura T. Takahashi N. Kato K. Arata Y. O-glycosylation in hinge region of mouse immunoglobulin G2b.J. Biolog. Chem. 1994; 269: 12345-12350Abstract Full Text PDF PubMed Google Scholar). Likewise, IgA1 was found to be more susceptible to degradation by Streptococci proteases after neuraminidase treatment (27Reinholdt J. Tomana M. Mortensen S.B. Kilian M. Molecular aspects of immunoglobulin A1 degradation by oral streptococci.Infection Immun. 1990; 58: 1186-1194Crossref PubMed Google Scholar). In this study, we report partial O-glycosylation of the human IgG3 hinge. We obtained both poly- and monoclonal IgG3 from various sources and performed proteolytic digestion with trypsin or proteinase K. NanoLC-reverse phase (RP)-ESI-ion trap (IT)-MS/MS was used to examine the resulting (glyco)peptides, revealing core 1-type O-glycans on multiple sites within the IgG3 hinge region. IgG3 was purified from six serum samples (average donor age: 44.5 ± 9 years; three male and three female; details are listed in supplemental Table S1). The serum samples were collected from healthy donors with informed consent in compliance with the institutional ethical board. Venous blood was collected in a 9-ml Vacuette serum clot activator tube (Greiner BioOne, Kremsminster, Austria) and incubated at room temperature for 30 min, followed by centrifugation for 15 min at 1800 g. The serum fraction was then collected and stored at −20 °C. IgG was isolated by running the serum over a HiTrap Protein G HP column (GE Healthcare, Buckinghamshire, UK) and eluted with 0.1 m glycine-HCl, pH 2.7. The eluate containing IgG was then applied to a HiTrap MabSelect SuRE column packed with recombinant Protein A (GE Healthcare), and the IgG3-containing flow-through was concentrated using an Amicon Ultra-15 centrifugal filter device 10 kDa (Merck Millipore, Darmstadt, Germany) and dialyzed against PBS using a Slide-A-Lizer Dialysis Cassette, 10K MWCO (Dionex/Thermo Scientific, Sunnyvale, CA). IgG3 purified from pooled plasma was obtained commercially from Athens Research and Technology (Athens, GA). Upon inquiry, we learned that this IgG3 had been treated with dextran sulfate, followed by centrifugation to pellet the lipids. The delipidated plasma was then subjected to an ammonium sulfate precipitation, followed by boric acid precipitation. The IgG was isolated with ion exchange chromatography, and further purification of IgG3 was achieved with affinity chromatography (rProtein A), gel filtration chromatography, and a jacalin column to remove a minor IgA contaminant. This purification method did not expose the IgG3 to extreme pH conditions or temperatures above 55 °C. Two monoclonal recombinant anti-GDob1 IgG3 allotypes, G3m(g) and G3m(s), were produced in an HEK-293F FreeStyle cell line expression system (Life Technologies, Paisley, UK). IgG3m(s) was then purified with a HiTrap MabSelect SuRE column packed with recombinant Protein A (GE Healthcare), while IgG3m(g) was purified with a Protein G HiTrap HP column (GE Healthcare). Three IgG3 G3m(g) hinge mutants were produced recombinantly, together with wild-type IgG3 G3m(g) as a control. The variable regions of the heavy and light chains (VH, VL) of the mouse IgG1 anti-2,4,6-trinitrophenol (TNP) hapten antibodies were cloned onto human IgG3 and kappa backbones, respectively, as described previously (20Kapur R. Kustiawan I. Vestrheim A. Koeleman C.A. Visser R. Einarsdottir H.K. Porcelijn L. Jackson D. Kumpel B. Deelder A.M. Blank D. Skogen B. Killie M.K. Michaelsen T.E. de Haas M. Rispens T. van der Schoot C.E. Wuhrer M. Vidarsson G. A prominent lack of IgG1-Fc fucosylation of platelet alloantibodies in pregnancy.Blood. 2014; 123: 471-480Crossref PubMed Scopus (145) Google Scholar, 28Kruijsen D. Einarsdottir H.K. Schijf M.A. Coenjaerts F.E. van der Schoot E.C. Vidarsson G. van Bleek G.M. Intranasal administration of antibody-bound respiratory syncytial virus particles efficiently primes virus-specific immune responses in mice.J. Virol. 2013; 87: 7550-7557Crossref PubMed Scopus (29) Google Scholar). Synthetic DNA encoding for IgG3 mutants, replacing the three threonines (T) and/or serines (S) with alanines (A), was generated as 5′-NheI and 3′Bsu36I fragments by GeneArt (Invitrogen). The NheI-Bsu36I fragments were ligated in anti-TNP IgG3 heavy chain replacing the corresponding fragment in the wild-type heavy chain. Antibodies were produced in the HEK-293F FreeStyle cell line expression system (Invitrogen) with cotransfection of vectors encoding p21, p27, and pSVLT genes as described (29Vink T. Oudshoorn-Dickmann M. Roza M. Reitsma J.J. de Jong R.N. A simple, robust and highly efficient transient expression system for producing antibodies.Methods. 2014; 65: 5-10Crossref PubMed Scopus (93) Google Scholar) to increase protein production. The antibodies were purified on a protein G HiTrap HP column (GE Healthcare) using the ÄKTAprime plus system (GE Healthcare) and dialyzed against PBS overnight. Four recombinant IgG3 and IgG4 Fc constructs were produced in a HEK-293F FreeStyle cell line (Invitrogen) and purified with a Protein G column (Protein G Sepharose 4 fast flow; GE Healthcare), as described by Rispens et al. (30Rispens T. Davies A.M. Ooijevaar-de Heer P. Absalah S. Bende O. Sutton B.J. Vidarsson G. Aalberse R.C. Dynamics of inter-heavy chain interactions in human immunoglobulin G (IgG) subclasses studied by kinetic Fab arm exchange.J. Biolog. Chem. 2014; 289: 6098-6109Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). All constructs contained an IgG4 hinge region, together with either the CH2 and CH3 regions of two IgG3 allotypes (G3m(b)-Fc-h4 and G3m(c3c5)-Fc-h4) or the CH2 and CH3 regions of two IgG4 variants (IgG4-Fc-V397M and IgG4-Fc-V397M,K392N). Several IgG4 samples were collected. A polyclonal IgG4 sample was affinity-purified from the plasma of a rheumatoid arthritis patient using anti-IgG4 coupled to Sepharose (clone MH164.4, Sanquin, Amsterdam, The Netherlands). A second IgG4 sample was enriched from the serum of a patient from the Leiden University Medical Center with an extremely high IgG4 serum titer (14.4 mg/ml). The serum was heat-inactivated and subjected to a 33% cut ammonium sulfate precipitation, followed by dialysis to decrease the salt content. The IgG4 was then enriched on a HiTrap DEAE Sepharose and a Superdex 200 column (GE Healthcare) using an ÄKTAprime plus chromatography system (GE Healthcare); IgG4-containing fractions were identified using ELISA and pooled. A monoclonal anti-TNP IgG4 sample produced in HEK cells was purified with a protein G column and dialyzed against PBS in the same way as described for the IgG3 mutants. In addition, we obtained a recombinant humanized IgG4 sample in the form of the therapeutic antibody natalizumab (Tysabri; Biogen Idec, Badehoevedorp, The Netherlands), which is produced in murine myeloma cells. The protein sequences of the recombinant samples can be found in supplemental Table S2. Five μg of the IgG samples was reduced with 2-beta-mercaptoethanol (Sigma-Aldrich, St. Louis, MO) at 95 °C for 10 min. The IgG was then run on a NuPage 4–12% Bis-Tris SDS-PAGE gel (Invitrogen) and stained with Coomassie G-250 (SimplyBlue SafeStain, Invitrogen). Bands were excised, cut into pieces, washed with 25 mm ammonium bicarbonate (ABC 1The abbreviations used are:ABCammonium bicarbonateC1qcomplement component 1 qCEcapillary electrophoresisCHconserved heavy chainCIDcollision-induced dissociationFabfragment antigen-bindingFcfragment crystallizableFcγRFc-gamma receptorGDob1chimeric MN12H2 antibodies with V genes from the human monoclonal IgG2 antibody DOB1HEKhuman embryonic kidneyHexhexoseHexNAcN-acetylhexosamineIgGimmunoglobulin gammaITion trapNeuAcN-acetylneuraminic acidt-ITPtransient isotachophoresisTNPtrinitrophenol., Sigma-Aldrich), and dehydrated with acetonitrile (Biosolve, Valkenswaard, The Netherlands). The IgG was then again treated with a reducing agent by adding 50 μl of a 10 mm dl-dithiothreitol (DTT; Sigma-Aldrich) 25 mm ABC solution for 30 min at 55 °C. The gel pieces were subsequently dehydrated by the addition of acetonitrile. Alkylation of the cysteine residues was achieved by incubation with 50 μl of a 55 mm iodoacetamide (Sigma-Aldrich) 25 mm ABC solution in the dark for 20 min. The gel pieces were then washed with 25 mm ABC and dehydrated with acetonitrile. The washing and dehydration was repeated a second time, and the samples were subsequently dried down completely in a centrifugal vacuum concentrator (Eppendorf, Hamburg, Germany). ammonium bicarbonate complement component 1 q capillary electrophoresis conserved heavy chain collision-induced dissociation fragment antigen-binding fragment crystallizable Fc-gamma receptor chimeric MN12H2 antibodies with V genes from the human monoclonal IgG2 antibody DOB1 human embryonic kidney hexose N-acetylhexosamine immunoglobulin gamma ion trap N-acetylneuraminic acid transient isotachophoresis trinitrophenol. For proteolytic digestion to take place, 30 μl of 25 mm ABC containing either trypsin (sequencing grade modified trypsin, Promega, Madison, WI), proteinase K (from Tritirachium album; Sigma-Aldrich) or chymotrypsin (sequencing grade from bovine pancreas, Roche Applied Sciences, Mannheim, Germany) was added to the dried gel pieces. An IgG:enzyme (w/w) ratio of 1:20 for trypsin, 1:3 for proteinase K and 1:20 for chymotrypsin was used. The samples were kept on ice for 1 h, to allow the enzyme to enter the gel pieces. If the gel pieces were not fully submerged, a further 10–20 μl of 25 mm ABC was added. The samples were incubated overnight at 37 °C. The solution surrounding the gel pieces was collected the next morning. Following the addition of another 20 μl of 25 mm ABC, the gel pieces were incubated at 37 °C for 1 h. The solution was then again collected and added to the first fraction, and stored at −20 °C. Alternatively, several IgG3 samples were digested in solution without reduction alkylation: 3 μg of IgG3 was incubated with 0.3 μg of trypsin in a total volume of 25 μl 25 mm ABC at 37 °C overnight. Endoproteinase AspN (New England Biolabs, Ipswich, MA) was used to further digest trypsin-generated (glyco)peptides. Digestion was performed by adding 1.5 μl of AspN and 15 μl of 2x AspN buffer (100 mm Tris-HCl, 5 mm zinc sulfate, New England Biolabs) to 15 μl of trypsin-digested IgG3, and incubating overnight at 37 °C. Exoglycosidase digestion was performed on tryptic IgG glycopeptides. The trypsin-digested IgG3 was first heated to 95 °C for 5 min to inactivate the trypsin. The sample was then dried in a centrifugal vacuum concentrator (Eppendorf), and resuspended by the addition of 16 μl of Milli-Q-purified water, 2 μl 50 mm sodium acetate (pH 5.5), 1 μl sialidase (Glyko sialidase A, Prozyme, Hayward, CA), and 1 μl of galactosidase (Glyko beta-galactosidase, Prozyme). The samples were incubated overnight at 37 °C. The IgG3 (glyco)peptides were analyzed with nanoLC-reversed phase (RP)-electrospray (ESI)-ion trap (IT)-MS(/MS) on an Ultimate 3000 RSLCnano system (Dionex/Thermo Scientific) coupled to an amaZon speed ESI-IT-MS (Bruker Daltonics, Bremen, Germany). A precolumn (Acclaim PepMap C18 capillary column, 300 μm x 5 mm, particle size 5 μm, Dionex/Thermo Scientific) was used to wash and concentrate the sample, and separation was achieved on an Acclaim PepMap RSLC C18 nanocolumn (75 μm x 150 mm, particle size 2 μm, Dionex/Thermo Scientific) with a flow rate of 500 nl/min. The following linear gradient was used, with solvent A consisting of 0.1% formic acid in water and solvent B of 95% acetonitrile, 5% water: t = 0 min, 1% solvent B; t = 5 min, 1% B; t = 20 min, 25% B; t = 25 min, 70% B; t = 30 min, 70% B; t = 31 min, 1% B; t = 55 min, 1% B. The sample was ionized in positive ion mode with an ESI-nanosprayer (4500 V) using a bare fused silica capillary (internal diameter of 20 μm). The solvent was evaporated at 180 °C with a nitrogen flow of 5 liters/min. A CaptiveSpray nanoBooster (Bruker Daltonics) was mounted onto the mass spectrometer and saturated the nitrogen flow with ACN to enhance the sensitivity. The MS1 ion detection window was set at m/z 350–1400, and the MS2 window at m/z 140–2200. The three highest nonsingly charged peaks in each MS1 spectrum were automatically fragmented through collision-induced dissociation (CID). In order to identify the peptide sequence of proteinase K- and trypsin-generated O-glycopeptides, MS3 analysis was performed on manually selected precursors: the MS2 peak representing the peptide without sugars attached was targeted for fragmentation. In a separate LC-MS run, electron transfer dissociation fragmentation was done on selected precursor ions. Transient isotachophoresis (ITP)-capillary electrophoresis (CE)-ESI-qTOF-MS/MS analysis was performed on a tryptic sialidase- and galactosidase-treated IgG3 sample derived from pooled plasma. Separation was achieved on a CESI 8000 system (AB Sciex, Framingham, MA), coupled via porous sheathless interfacing to an ultra-high resolution (UHR)-qTOF maXis Impact MS system (Bruker
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