N- and O-glycosylation Analysis of Human C1-inhibitor Reveals Extensive Mucin-type O-Glycosylation
2017; Elsevier BV; Volume: 17; Issue: 6 Linguagem: Inglês
10.1074/mcp.ra117.000240
ISSN1535-9484
AutoresKathrin Stavenhagen, Hacı Mehmet Kayılı, Stephanie Holst, Carolien A. M. Koeleman, Ruchira Engel, Diana Wouters, Sacha Zeerleder, Bekir Salih, Manfred Wuhrer,
Tópico(s)Peptidase Inhibition and Analysis
ResumoHuman C1-inhibitor (C1-Inh) is a serine protease inhibitor and the major regulator of the contact activation pathway as well as the classical and lectin complement pathways. It is known to be a highly glycosylated plasma glycoprotein. However, both the structural features and biological role of C1-Inh glycosylation are largely unknown. Here, we performed for the first time an in-depth site-specific N- and O-glycosylation analysis of C1-Inh combining various mass spectrometric approaches, including C18-porous graphitized carbon (PGC)-LC-ESI-QTOF-MS/MS applying stepping-energy collision-induced dissociation (CID) and electron-transfer dissociation (ETD). Various proteases were applied, partly in combination with PNGase F and exoglycosidase treatment, in order to analyze the (glyco)peptides. The analysis revealed an extensively O-glycosylated N-terminal region. Five novel and five known O-glycosylation sites were identified, carrying mainly core1-type O-glycans. In addition, we detected a heavily O-glycosylated portion spanning from Thr82-Ser121 with up to 16 O-glycans attached. Likewise, all known six N-glycosylation sites were covered and confirmed by this site-specific glycosylation analysis. The glycoforms were in accordance with results on released N-glycans by MALDI-TOF/TOF-MS/MS. The comprehensive characterization of C1-Inh glycosylation described in this study will form the basis for further functional studies on the role of these glycan modifications. Human C1-inhibitor (C1-Inh) is a serine protease inhibitor and the major regulator of the contact activation pathway as well as the classical and lectin complement pathways. It is known to be a highly glycosylated plasma glycoprotein. However, both the structural features and biological role of C1-Inh glycosylation are largely unknown. Here, we performed for the first time an in-depth site-specific N- and O-glycosylation analysis of C1-Inh combining various mass spectrometric approaches, including C18-porous graphitized carbon (PGC)-LC-ESI-QTOF-MS/MS applying stepping-energy collision-induced dissociation (CID) and electron-transfer dissociation (ETD). Various proteases were applied, partly in combination with PNGase F and exoglycosidase treatment, in order to analyze the (glyco)peptides. The analysis revealed an extensively O-glycosylated N-terminal region. Five novel and five known O-glycosylation sites were identified, carrying mainly core1-type O-glycans. In addition, we detected a heavily O-glycosylated portion spanning from Thr82-Ser121 with up to 16 O-glycans attached. Likewise, all known six N-glycosylation sites were covered and confirmed by this site-specific glycosylation analysis. The glycoforms were in accordance with results on released N-glycans by MALDI-TOF/TOF-MS/MS. The comprehensive characterization of C1-Inh glycosylation described in this study will form the basis for further functional studies on the role of these glycan modifications. Human C1-inhibitor (C1-Inh) 1The abbreviations used are: AmBiC, ammonium bicarbonate; C1-Inh, C1-Inhibitor; ENeuAc, α2,6-linked N-acetylneuraminic acid; EDC, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride; ETD, electron-transfer dissociation; FA, formic acid; Fuc, fucose; GalNAc, N-acetylgalactosamine; GlcNAc, N-acetylglucosamine; Hereditary angioedema, HAE; Hex, hexose; HexNAc, N-acetylhexosamine; HILIC, hydrophilic interaction liquid chromatography; HOBt, hydroxybenzotriazol hydrate; IAA, iodoacetamide; IPQ score, isotopic pattern quality score; IT, ion trap; LNeuAc, α2,3-linked N-acetylneuraminic acid; Na2HPO4x2H2O, sodium hydrogen phosphate dihydrate; NaOH, sodium hydroxide; NeuAc, N-acetylneuraminic acid; NP-40, Nonidet P-40; PGC, porous graphitized carbon; PNGase F, peptide-N-glycosidase F; RP, reversed-phase; sDHB, 2-hydroxy-5-methoxy-benzoic acid and 2,5-dihydroxybenzoic acid; Serpin, serine protease inhibitor; SPE, solid phase extraction. 1The abbreviations used are: AmBiC, ammonium bicarbonate; C1-Inh, C1-Inhibitor; ENeuAc, α2,6-linked N-acetylneuraminic acid; EDC, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride; ETD, electron-transfer dissociation; FA, formic acid; Fuc, fucose; GalNAc, N-acetylgalactosamine; GlcNAc, N-acetylglucosamine; Hereditary angioedema, HAE; Hex, hexose; HexNAc, N-acetylhexosamine; HILIC, hydrophilic interaction liquid chromatography; HOBt, hydroxybenzotriazol hydrate; IAA, iodoacetamide; IPQ score, isotopic pattern quality score; IT, ion trap; LNeuAc, α2,3-linked N-acetylneuraminic acid; Na2HPO4x2H2O, sodium hydrogen phosphate dihydrate; NaOH, sodium hydroxide; NeuAc, N-acetylneuraminic acid; NP-40, Nonidet P-40; PGC, porous graphitized carbon; PNGase F, peptide-N-glycosidase F; RP, reversed-phase; sDHB, 2-hydroxy-5-methoxy-benzoic acid and 2,5-dihydroxybenzoic acid; Serpin, serine protease inhibitor; SPE, solid phase extraction. is a serine protease inhibitor (serpin) and the major regulator of the contact activation pathway via inhibition of factor XIIa, kallikrein and factor XIa, as well as the classical and lectin complement pathways via C1s, C1r, and MASP (1.Caliezi C. Wuillemin W.A. Zeerleder S. Redondo M. Eisele B. Hack C.E. C1-esterase inhibitor : an anti-inflammatory agent and its potential use in the treatment of diseases other than hereditary angioedema.Pharmacol. Rev. 2000; 52: 91-112PubMed Google Scholar). A C1-Inh deficiency, either because of decreased or dysfunctional expression, is associated with hereditary angioedema (HAE), which results in vascular permeability causing tissue swelling (1.Caliezi C. Wuillemin W.A. Zeerleder S. Redondo M. Eisele B. Hack C.E. C1-esterase inhibitor : an anti-inflammatory agent and its potential use in the treatment of diseases other than hereditary angioedema.Pharmacol. Rev. 2000; 52: 91-112PubMed Google Scholar). For treatment and prophylaxis of HAE human plasma-derived C1-Inh replacement therapy is commonly applied (2.Bowen T. Cicardi M. Farkas H. Longhurst H.J. Zuraw B. Aygoeren-Pürsün E. Craig T. Binkley K. Hebert J. Ritchie B. Bouillet L. Betschel S. Cogar D. Dean J. Devaraj R. Hamed A. Kamra P. Keith P.K. Lacuesta G. Leith E. Lyons H. Mace S. Mako B. Neurath D. Poon M.C. Rivard G.E. Schellenberg R. Rowan D. Rowe A. Stark D. Sur S. Tsai E. Warrington R. Waserman S. Ameratunga R. Bernstein J. Björkander J. Brosz K. Brosz J. Bygum A. Caballero T. Frank M. Fust G. Harmat G. Kanani A. Kreuz W. Levi M. Li H. Martinez-Saguer I. Moldovan D. Nagy I. Nielsen E.W. Nordenfelt P. Reshef A. Rusicke E. Smith-Foltz S. Späth P. Varga L. Xiang Z.Y. 2010 International consensus algorithm for the diagnosis, therapy and management of hereditary angioedema.Allergy, Asthma, Clin. Immunol. 2010; 6: 24Crossref PubMed Google Scholar). Also other diseases, such as inflammatory diseases, sepsis and endotoxic shock, may be targeted by C1-Inh therapy (1.Caliezi C. Wuillemin W.A. Zeerleder S. Redondo M. Eisele B. Hack C.E. C1-esterase inhibitor : an anti-inflammatory agent and its potential use in the treatment of diseases other than hereditary angioedema.Pharmacol. Rev. 2000; 52: 91-112PubMed Google Scholar, 3.Davis A.E. Lu F. Mejia P. C1 inhibitor, a multi-functional serine protease inhibitor.Thromb. Haemost. 2010; 104: 886-893Crossref PubMed Scopus (164) Google Scholar). To meet this need, recombinant C1-Inh formats are currently being developed (4.Koles K. van Berkel P.H.C. Mannesse M.L.M. Zoetemelk R. Vliegenthart J.F. Kamerling J.P. Influence of lactation parameters on the N-glycosylation of recombinant human C1 inhibitor isolated from the milk of transgenic rabbits.Glycobiology. 2004; 14: 979-986Crossref PubMed Scopus (27) Google Scholar, 5.Koles K. van Berkel P.H.C. Pieper F.R. Nuijens J.H. Mannesse M.L. Vliegenthart J.F. Kamerling J.P. N- and O-glycans of recombinant human C1 inhibitor expressed in the milk of transgenic rabbits.Glycobiology. 2004; 14: 51-64Crossref PubMed Scopus (93) Google Scholar, 6.Bos I.G.A. De Bruin E.C. Karuntu Y.A. Modderman P.W. Eldering E. Hack C.E. Recombinant human C1-inhibitor produced in Pichia pastoris has the same inhibitory capacity as plasma C1-inhibitor.Biochim Biophys Acta. 2003; 1648: 75-83Crossref PubMed Scopus (27) Google Scholar, 7.Lamark T. Ingebrigtsen M. Bjørnstad C. Melkko T. Mollnes T.E. Nielsen E.W. Expression of active human C1 inhibitor serpin domain in Escherichia coli.Protein Expr. Purif. 2001; 22: 349-358Crossref PubMed Scopus (36) Google Scholar). C1-Inh is considered as one of the most heavily glycosylated proteins in human plasma (1.Caliezi C. Wuillemin W.A. Zeerleder S. Redondo M. Eisele B. Hack C.E. C1-esterase inhibitor : an anti-inflammatory agent and its potential use in the treatment of diseases other than hereditary angioedema.Pharmacol. Rev. 2000; 52: 91-112PubMed Google Scholar). The protein consists of 478 amino acids and the calculated molecular mass of C1-Inh is ∼53 kDa without glycans, whereas a much higher apparent molecular mass was observed on SDS-PAGE (>80kDa) because of its heavy glycosylation (8.Perkins S.J. Smith K.F. Amatayakul S. Ashford D. Rademacher T.W. Dwek R.A. Lachmann P.J. Harrison R.A. Two-domain structure of the native and reactive centre cleaved forms of C1 inhibitor of human complement by neutron scattering.J. Mol. Biol. 1990; 214: 751-763Crossref PubMed Scopus (48) Google Scholar, 9.Nuijens J.H. Eerenberg-Belmer J.M. Huijbregts C.C.M. Schreuder W.O. Felt-Bersma R.J. Abbink J.J. Thijs L.G. Hack C.E. Proteolytic inactivation of plasma C1 inhibitor in sepsis.J. Clin. Invest. 1989; 84: 443-450Crossref PubMed Scopus (115) Google Scholar). It has been reported that C1-Inh possesses six occupied N- and up to 24 O-glycosylation sites (8.Perkins S.J. Smith K.F. Amatayakul S. Ashford D. Rademacher T.W. Dwek R.A. Lachmann P.J. Harrison R.A. Two-domain structure of the native and reactive centre cleaved forms of C1 inhibitor of human complement by neutron scattering.J. Mol. Biol. 1990; 214: 751-763Crossref PubMed Scopus (48) Google Scholar, 10.Halim A. Rüetschi U. Larson G. Nilsson J. LC-MS/MS characterization of O-glycosylation sites and glycan structures of human cerebrospinal fluid glycoproteins.J. Proteome Res. 2013; 12: 573-584Crossref PubMed Scopus (90) Google Scholar, 11.Bock S.C. Skriver K. Nielsen E. et al.Human C1 inhibitor: primary structure, cDNA cloning, and chromosomal localization.Biochemistry. 1986; 25: 4292-4301Crossref PubMed Scopus (268) Google Scholar, 12.Strecker G. Ollierhartmann M.P. Vanhalbeek H. et al.Primary structure elucidation of carbohydrate chains of normal c1-esterase inhibitor (C1-Inh) by 400-Mhz H-1-NMR study.C. R. Acad. Sc. Paris. 1985; 301: 571-576PubMed Google Scholar, 13.King S.L. Joshi H.J. Schjoldager K.T. Halim A. Madsen T.D. Dziegiel M.H. Woetmann A. Vakhrushev S.Y. Wandall H.H. Characterizing the O-glycosylation landscape of human plasma, platelets, and endothelial cells.Blood Adv. 2017; 1: 429-442Crossref PubMed Scopus (96) Google Scholar). Of the latter ones ten have been identified with their exact location (10.Halim A. Rüetschi U. Larson G. Nilsson J. LC-MS/MS characterization of O-glycosylation sites and glycan structures of human cerebrospinal fluid glycoproteins.J. Proteome Res. 2013; 12: 573-584Crossref PubMed Scopus (90) Google Scholar, 11.Bock S.C. Skriver K. Nielsen E. et al.Human C1 inhibitor: primary structure, cDNA cloning, and chromosomal localization.Biochemistry. 1986; 25: 4292-4301Crossref PubMed Scopus (268) Google Scholar, 13.King S.L. Joshi H.J. Schjoldager K.T. Halim A. Madsen T.D. Dziegiel M.H. Woetmann A. Vakhrushev S.Y. Wandall H.H. Characterizing the O-glycosylation landscape of human plasma, platelets, and endothelial cells.Blood Adv. 2017; 1: 429-442Crossref PubMed Scopus (96) Google Scholar). The protein consists of two domains: (1) the C-terminal domain (serpin domain), which carries three of the six N-glycosylation sites, provides the inhibition activity of C1-Inh and is similar to other serpins; and (2) the N-terminal domain, which consists of ∼135–142 amino acid residues (∼113–120 amino acids in the mature protein), featuring the remaining three N- and all O-glycosylation sites (11.Bock S.C. Skriver K. Nielsen E. et al.Human C1 inhibitor: primary structure, cDNA cloning, and chromosomal localization.Biochemistry. 1986; 25: 4292-4301Crossref PubMed Scopus (268) Google Scholar, 14.Perkins S.J. Three-dimensional structure and molecular modelling of C1- inhibitor.Behring Inst. Mitt. 1993; 93: 63-80PubMed Google Scholar). Even though protein glycosylation has a large impact on biological processes, protein stability, and protein functions (15.Dwek R.A. Glycobiology: toward understanding the function of sugars.Chem. Rev. 1996; 96: 683-720Crossref PubMed Scopus (2840) Google Scholar, 16.Bieberich E. Synthesis, processing, and function of N-glycans in N- glycoproteins.Adv. Neurobiol. 2014; 9: 47-70Crossref PubMed Google Scholar), the structural features as well as biological role of C1-Inh glycosylation is still largely unknown. To address this, we here present a detailed site-specific N- and O-glycosylation characterization of plasma derived C1-Inh using a panel of mass spectrometric approaches. The C1-Inh glycosylation as studied here will inform further functional studies in order to understand glycan involvement in C1-Inh function. Furthermore, this plasma-derived human C1-Inh glycosylation will serve as a benchmark for evaluating the glycosylation profiles of recombinant C1-Inh. Ammonium bicarbonate (AmBiC), hydroxybenzotriazole hydrate (HOBt), iodoacetamide (IAA), 2-mercaptoethanol, Nonidet P-40 (NP-40), PBS, formic acid (FA), sDHB (2-hydroxy-5-methoxy-benzoic acid and 2,5-dihydroxybenzoic acid, 1:9) as well as a 50% sodium hydroxide (NaOH) solution were obtained from Sigma-Aldrich (Steinheim, Germany). Sodium hydrogen phosphate dihydrate (Na2HPO4x2H2O), sodium bicarbonate, DTT, ethanol, potassium dihydrogen phosphate, SDS, sodium chloride and TFA were purchased from Merck (Darmstadt, Germany). 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) originated from Fluorochem (Hadfield, UK) and HPLC SupraGradient ACN from Biosolve (Valkenswaard, The Netherlands). Commercial C1-Inh (Cetor®), isolated from pooled cryo- and 4F-depleted plasma, using ion exchange chromatography and PEG precipitation, was obtained from Sanquin, Amsterdam, The Netherlands. For samples to be treated with exoglycosidases, de-N-glycosylation was achieved using an in-solution Peptide-N-glycosidase F (PNGase F; Roche Diagnostics, Mannheim, Germany) approach prior to in-gel digestion with proteolytic enzymes. Ten microliter C1-Inh (1 μg/μl) were first denatured and reduced with a mixture of 1 μl of 5% SDS and 0.4 m DTT by shaking thoroughly for 5 min at 95 °C. Then, 10 μl PNGase F solution (1 U in 2% NP-40/2 x PBS) was added, followed by an overnight incubation at 37 °C. De-N-glycosylated C1-Inh was then subjected to SDS-PAGE and in-gel proteolytic digestion as described below. In-gel protease digestion (10 μg protein) of C1-Inh (supplemental Fig. S1), was performed as previously described (17.Plomp R. Hensbergen P.J. Rombouts Y. Zauner G. Dragan I. Koeleman C.A. Deelder A.M. Wuhrer M. Site-specific N-glycosylation analysis of human immunoglobulin E.J. Proteome Res. 2014; 13: 536-546Crossref PubMed Scopus (69) Google Scholar) using either 0.15 μg of trypsin (sequencing grade; Promega, Madison, WI), 1 μg of Proteinase K (Tritirachium album, Sigma-Aldrich) or 1 μg Pronase (Streptomyces griseus, Sigma-Aldrich) in 30 μl of 25 mm AmBiC. De-N-glycosylated glycopeptides were obtained by in-gel PNGase F treatment (details in released N-glycan section) followed by in-gel proteolytic digestion, except for glycopeptide samples that were further treated with exoglycosidases (see in-solution PNGase F treatment). After in-gel PNGase F treatment the gel pieces were washed twice for 5 min with 100 μl 25 mm AmBiC before in-gel proteolytic digestion. (Glyco)-peptides were dried in a centrifugal vacuum concentrator and reconstituted by adding 16 μl of water, 2 μl sodium acetate (50 mm, pH 5.5), 1 μl sialidase (5 mU, Glyko sialidase A; Prozyme, Hayward, CA), and 1 μl of galactosidase (5 mU, Glyko beta-galactosidase, Prozyme). The digestions were carried out overnight at 37 °C. C18-porous graphitized carbon (PGC)-LC-ESI-QTOF-MS/MS analysis was performed as described previously (18.Stavenhagen K. Plomp R. Wuhrer M. Site-specific protein N- and O-glycosylation analysis by a C18-porous graphitized carbon-liquid chromatography-electrospray ionization mass spectrometry approach using Pronase treated glycopeptides.Anal. Chem. 2015; 87: 11691-11699Crossref PubMed Scopus (58) Google Scholar) using a maXis HD QTOF mass spectrometer equipped with a CaptiveSpray nanoBooster source (both Bruker Daltonics) coupled to an Ultimate 3000 × 2 dual analytical nanoUPLC system (Thermo Scientific). The LC-MS setup controlled by Hystar 3.2 (Bruker Daltonics) and data analysis was performed using DataAnalysis 4.2 (Bruker Daltonics). A combined C18-PGC-LC approach was applied to separate Pronase- and Proteinase K- treated (glyco)peptides (18.Stavenhagen K. Plomp R. Wuhrer M. Site-specific protein N- and O-glycosylation analysis by a C18-porous graphitized carbon-liquid chromatography-electrospray ionization mass spectrometry approach using Pronase treated glycopeptides.Anal. Chem. 2015; 87: 11691-11699Crossref PubMed Scopus (58) Google Scholar). The two valve nanoUPLC system was used with the following setup: valve 1 was equipped with a C18 precolumn (C18 PepMap 100, 300 μm x 5 mm, 5 μm, 100 Å, Thermo Scientific) and analytical column (Acclaim PepMap RSLC, 75 μm × 15 cm, 2 μm, 100 Å; Thermo Scientific) and valve 2 with a PGC precolumn (in-house made, 100 μm × 15 mm, 3 μm Hypercarb material; Thermo Scientific) and analytical column (in-house made, 50 μm × 150 mm, 3 μm Hypercarb material; Thermo Scientific). During loading, both precolumns were switched in-line allowing the sample first to pass the C18 precolumn and then to directly load the flow-through, with all unbound compounds, onto the PGC precolumn. In a second step the valves were switched for sequential elution of the compounds from the two precolumns over their corresponding analytical columns. A post-column nano valve directed the flow of the C18 or PGC column system subsequently to the mass spectrometer. Pronase- and Proteinase K-treated glycopeptides were diluted 10 times and 4 μl were loaded onto the precolumns with loading solvent (99% water/1% ACN/0.05% TFA) at a flow rate of 6 μl/min and column oven temperature of 36 °C. The C18-PGC-LC setup was operated with solvent A (water containing 0.1% FA (v/v)) and solvent B (80% acetonitrile/20% water containing 0.1% FA (v/v)). First, (glyco)peptides from the C18 columns were eluted with a flow rate of 500 nL/min using a linear gradient (t = 5–35 min, c(B) = 1–55%), followed by column washing and reconditioning. After 28 min a post-column nano-valve was switched and the flow from the PGC columns was sent to the MS. The elution of the PGC columns was performed with a linear gradient (t = 22–55 min, c(B) = 1–40%) at a flow rate of 400 nL/min, followed by column washing and reconditioning. Ionization was enhanced using a nanoBooster (Bruker Daltonics) with acetonitrile-enriched nitrogen at 0.2 bar. The source parameters were set to a dry gas flow of 3 L/min at 150 °C and a capillary voltage of 1200 V. The mass spectrometer was calibrated using ESI-l-low concentration tuning mixture (Agilent Technologies, Santa Clara, CA). MS acquisition was performed within a mass range of m/z 50 to m/z 2800 at a spectra rate of 1 Hz. Basic stepping mode was applied for the MS/MS collision energy (80 and 140%) each for 50% of the time. Collision energies were set as follows: For singly charged precursors 45 eV at m/z 500, 60 eV at m/z 800, 80 eV at m/z 1300; for doubly charged precursors 25 eV at m/z 500, 47 eV at m/z 800, 60 eV at m/z 1300, for precursors with three and more charges 20 eV at m/z 500, 45 eV at m/z 800, 65 eV at m/z 1300. MS/MS was performed on the three most abundant precursor ions at a spectra acquisition rate of 0.5 Hz to 2 Hz depending on the precursor intensity (18.Stavenhagen K. Plomp R. Wuhrer M. Site-specific protein N- and O-glycosylation analysis by a C18-porous graphitized carbon-liquid chromatography-electrospray ionization mass spectrometry approach using Pronase treated glycopeptides.Anal. Chem. 2015; 87: 11691-11699Crossref PubMed Scopus (58) Google Scholar). C18-RP-LC-ESI-QTOF-MS/MS analysis was performed on the same LC-MS system as used for C18-PGC-LC-ESI-QTOF-MS/MS. All trypsin-treated samples were diluted 30 times with water and 5 μl were loaded onto a C18 precolumn (C18 PepMap 100, 100 μm x 2 cm, 5 μm, 100 Å, Dionex/Thermo Scientific, Breda, The Netherlands) with 15 μl/min of loading solvent (water containing 0.1% ACN/0.1% FA) for 2 min. The analytes were separated on a C18 analytical column (Acclaim PepMap RSLC, 75 μm × 15 cm, 2 μm, 100 Å, Dionex/Thermo Scientific) at 32 °C column oven temperature. Elution was performed at a flow rate of 0.7 μl/min with solvent A (water containing 0.1% formic acid (FA) (v/v)) and solvent B (95% acetonitrile (ACN)/5% water containing 0.1% FA (v/v)). A linear gradient of 3–31.7% solvent B in 25 min was applied followed by column washing and reconditioning. The MS was operated in stepping-energy CID mode as described previously (setup two in (19.Hinneburg H. Stavenhagen K. Schweiger-Hufnagel U. Pengelley S. Jabs W. Seeberger P.H. Silva D.V. Wuhrer M. Kolarich D. The art of destruction: optimizing collision energies in quadrupole-time of flight (Q-TOF) instruments for glycopeptide-based glycoproteomics.J. Am. Soc. Mass Spectrom. 2016; 27: 507-519Crossref PubMed Scopus (90) Google Scholar)). To acquire data for relative quantitation the MS was operated in MS only mode. For electron-transfer dissociation (ETD) experiments the MS parameters were set as described for stepping-energy CID, except of the collision RF, which was set to 500 and 800 Vpp in basic stepping mode (each 50% of the time). The ICC target was set to 3 Mio for an accumulation time of max. 600 ms. The reagent injection was 45 ms and the extended reaction time 5 ms. ETD precursors were selected using a target list obtained from CID runs. DataAnalysis 4.2 software (Bruker Daltonics) was used to analyze glycopeptides of C1-Inh (P05155) by manually scanning for glycan oxonium ions. The defined stepping-energy CID glycopeptide spectra were analyzed manually to identify the glycan structure and the mass of the peptide backbone as described previously (18.Stavenhagen K. Plomp R. Wuhrer M. Site-specific protein N- and O-glycosylation analysis by a C18-porous graphitized carbon-liquid chromatography-electrospray ionization mass spectrometry approach using Pronase treated glycopeptides.Anal. Chem. 2015; 87: 11691-11699Crossref PubMed Scopus (58) Google Scholar), including carbamidomethylation as a fixed and oxidation as a variable modification. Additionally, also lower mass range oxonium ions of stepping-energy CID spectra were used to characterize the glycan portion. The ratio of HexNAc fragments in higher-energy CID can be a marker for the presence of GlcNAc in the glycopeptide (20.Halim A. Westerlind U. Pett C. Schorlemer M. Rüetschi U. Brinkmalm G. Sihlbom C. Lengqvist J. Larson G. Nilsson J. Assignment of saccharide identities through analysis of oxonium ion fragmentation profiles in LC-MS/MS of glycopeptides.J. Proteome Res. 2014; 13: 6024-6032Crossref PubMed Scopus (100) Google Scholar). A high ratio of m/z 138 ([HexNAc-CH6O3]+) + m/z 168 ([HexNAc-2H2O]+) relative to m/z 126 ([HexNAc-C2H6O3]+) + m/z 144 ([HexNAc-C2H4O2]+) is diagnostic for a GlcNAc-containing glycopeptide, close to equal intensities of m/z 138 + m/z 168 compared with m/z 126 + m/z 144 are indicative for only GalNAc-containing glycopeptides. For a selected list of identified glycopeptides with known compositions and retention times, based on CID spectra, also ETD spectra were manually analyzed, allowing a mass deviation of 10 ppm (QTOF) and 0.1 Da (IT). For relative quantitation signal intensities of all tryptic glycopeptides and partially also of miss-cleaved ones (up to one miss cleavage) were extracted in an automated manner using LaCy tools (version 1.0.0) (21.Jansen B.C. Falck D. de Haan N. Hipgrave Ederveen A.L. Razdorov G. Lauc G. Wuhrer M. LaCyTools – a targeted LC-MS data processing package for relative quantitation of glycopeptides.J. Proteome Res. 2016; 15: 2198-2210Crossref PubMed Scopus (78) Google Scholar). LaCy tools settings were as follows: sum spectrum resolution = 100; mass window 0.07 Th; time window 18 s; minimum percentage of the total theoretical isotopic distribution = 95%, background window = 10 Th. The analyte was included for relative quantitation based on the following criteria: signal-to-noise of at least 9; average mass error of ±10 ppm, average isotopic pattern quality (IPQ) score ≤0.25. The samples were analyzed in triplicates. The data was normalized based on the total intensity of all compounds and the standard deviation was calculated. In-gel N-glycan release (10 μg protein) was performed as previously described (17.Plomp R. Hensbergen P.J. Rombouts Y. Zauner G. Dragan I. Koeleman C.A. Deelder A.M. Wuhrer M. Site-specific N-glycosylation analysis of human immunoglobulin E.J. Proteome Res. 2014; 13: 536-546Crossref PubMed Scopus (69) Google Scholar) with minor modifications. Different from the protocol, the gel bands were washed with 25 mm sodium bicarbonate, pH 8, instead of AmBiC. For the N-glycan release, 20 μl to 30 μl PNGase F solution (2 U (Roche Diagnostics) in 2% Nonidet P-40 (NP-40) and 2.5xPBS) were used. Released N-glycans were derivatized by ethyl esterification (22.Reiding K.R. Blank D. Kuijper D.M. Deelder A.M. Wuhrer M. High-throughput profiling of protein N-glycosylation by MALDI-TOF-MS employing linkage-specific sialic acid esterification.Anal. Chem. 2014; 86: 5784-5793Crossref PubMed Scopus (253) Google Scholar) followed by glycan purification by hydrophilic interaction chromatography (HILIC)-solid phase extraction (SPE) using cotton thread modified from a protocol described previously (23.Selman M.H.J. Hemayatkar M. Deelder A.M. Wuhrer M. Cotton HILIC SPE microtips for microscale purification and enrichment of glycans and glycopeptides.Anal. Chem. 2011; 83: 2492-2499Crossref PubMed Scopus (251) Google Scholar). The N-glycans were eluted in 10 μl water. From this, 5 μl were used for mass spectrometric analysis by spotting them onto an anchor chip matrix-assisted laser desorption dissociation (MALDI) target plate (Bruker Daltonics, Bremen, Germany) and cocrystallized with 1 μl of 5 mg/ml sDHB (2-hydroxy-5-methoxy-benzoic acid and 2,5-dihydroxybenzoic acid, 1:9, Sigma-Aldrich) in 50% ACN/50% water containing 1 mm NaOH. MALDI-TOF-MS spectra were acquired using an UltrafleXtreme mass spectrometer (Bruker Daltonics) in positive ion reflector mode. Spectra were obtained over a mass window of m/z 1000 to m/z 5000 with suppression up to m/z 900 for a total of 20,000 shots. Tandem mass spectrometry (MALDI-TOF/TOF-MS/MS) was performed for structural elucidation via fragmentation in gas-off TOF/TOF mode. A compound list of C1-Inh N-glycans was manually curated and relative quantitation of the N-glycoforms was performed using an in-house developed software for automated data processing MassyTools 1.0 (24.Jansen B.C. Reiding K.R. Bondt A. Hipgrave Ederveen A.L. Palmblad M. Falck D. Wuhrer M. MassyTools: A high-throughput targeted data processing tool for relative quantitation and quality control developed for glycomic and glycoproteomic MALDI-MS.J. Proteome Res. 2015; 14: 5088-5098Crossref PubMed Scopus (77) Google Scholar). Only glycan compositions that have been confirmed by MS/MS and their directly related compositions (± one monosaccharide) were taken into account for relative quantitation. Detailed information of the released N-glycan sample preparation and analysis is provided in the supplemental data. A detailed N- and O-glycosylation analysis of C1-Inh was performed using various MS-based approaches. C1-Inh glycopeptides were generated by subjecting the purified glycoprotein to various protease treatments with and without the addition of PNGase F and exoglycosidases for N-glycan release and glycan trimming, respectively. This resulted in N-, O- and N-/O-glycopeptides of different complexity to achieve a high glycopeptide coverage. Additionally, C1-Inh N-glycans were released and analyzed. Sample preparation, followed by MS analysis was performed in triplicates (glycopeptide analysis) or quadruplicates (N-glycan analysis) and the average relative distribution of glycopeptides, N-glycans and their respective standard deviations were calculated. For O-glycosylation site identification and characterization C1-Inh was subjected to in-gel trypsin, Pronase, and Proteinase K treatment. The latter two broad-specificity proteases commonly result in smaller peptide portions to reduce sample complexity for tandem MS analysis. To further decrease sample heterogeneity and enhance O-glycosylation site identification a portion of these digests were de-N-glycosylated by PNGase F and partially also processed with exoglycosidases such as sialidase and galactosidase. This approach aimed to trim down short mucin-type O-glycans, to obtain O-glycopeptides with a single N-acetylhexosamine (HexNAc) or HexNAc-hexose (Hex) moiety attached to the O-glycosylation site, which allowed a more reliable site-specific analysis. Samples were analyzed by nanoLC-MS/MS analysis applying different tandem MS modes to obtain more structural information of the glycopeptides. For this, we applied a C18-PGC-LC-ESI-QTOF-MS/MS approach that has recently been developed by us and facilitates higher glycopeptide coverage after Pronase and Proteinase K treatme
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