Flagellin Glycoproteomics of the Periodontitis Associated Pathogen Selenomonas sputigena Reveals Previously Not Described O-glycans and Rhamnose Fragment Rearrangement Occurring on the Glycopeptides
2018; Elsevier BV; Volume: 17; Issue: 4 Linguagem: Inglês
10.1074/mcp.ra117.000394
ISSN1535-9484
AutoresCornelia B. Rath, Falko Schirmeister, Rudolf Figl, Peter H. Seeberger, Christina Schäffer, Daniel Kolarich,
Tópico(s)Genomics and Phylogenetic Studies
ResumoFlagellated, Gram-negative, anaerobic, crescent-shaped Selenomonas species are colonizers of the digestive system, where they act at the interface between health and disease. Selenomonas sputigena is also considered a potential human periodontal pathogen, but information on its virulence factors and underlying pathogenicity mechanisms is scarce. Here we provide the first report of a Selenomonas glycoprotein, showing that S. sputigena produces a diversely and heavily O-glycosylated flagellin C9LY14 as a major cellular protein, which carries various hitherto undescribed rhamnose- and N-acetylglucosamine linked O-glycans in the range from mono- to hexasaccharides. A comprehensive glycomic and glycoproteomic assessment revealed extensive glycan macro- and microheterogeneity identified from 22 unique glycopeptide species. From the multiple sites of glycosylation, five were unambiguously identified on the 437-amino acid C9LY14 protein (Thr149, Ser182, Thr199, Thr259, and Ser334), the only flagellin protein identified. The O-glycans additionally showed modifications by methylation and putative acetylation. Some O-glycans carried hitherto undescribed residues/modifications as determined by their respective m/z values, reflecting the high diversity of native S. sputigena flagellin. We also found that monosaccharide rearrangement occurred during collision-induced dissociation (CID) of protonated glycopeptide ions. This effect resulted in pseudo Y1-glycopeptide fragment ions that indicated the presence of additional glycosylation sites on a single glycopeptide. CID oxonium ions and electron transfer dissociation, however, confirmed that just a single site was glycosylated, showing that glycan-to-peptide rearrangement can occur on glycopeptides and that this effect is influenced by the molecular nature of the glycan moiety. This effect was most pronounced with disaccharides. This study is the first report on O-linked flagellin glycosylation in a Selenomonas species, revealing that C9LY14 is one of the most heavily glycosylated flagellins described to date. This study contributes to our understanding of the largely under-investigated surface properties of oral bacteria. The data have been deposited to the ProteomeXchange with identifier PXD005859. Flagellated, Gram-negative, anaerobic, crescent-shaped Selenomonas species are colonizers of the digestive system, where they act at the interface between health and disease. Selenomonas sputigena is also considered a potential human periodontal pathogen, but information on its virulence factors and underlying pathogenicity mechanisms is scarce. Here we provide the first report of a Selenomonas glycoprotein, showing that S. sputigena produces a diversely and heavily O-glycosylated flagellin C9LY14 as a major cellular protein, which carries various hitherto undescribed rhamnose- and N-acetylglucosamine linked O-glycans in the range from mono- to hexasaccharides. A comprehensive glycomic and glycoproteomic assessment revealed extensive glycan macro- and microheterogeneity identified from 22 unique glycopeptide species. From the multiple sites of glycosylation, five were unambiguously identified on the 437-amino acid C9LY14 protein (Thr149, Ser182, Thr199, Thr259, and Ser334), the only flagellin protein identified. The O-glycans additionally showed modifications by methylation and putative acetylation. Some O-glycans carried hitherto undescribed residues/modifications as determined by their respective m/z values, reflecting the high diversity of native S. sputigena flagellin. We also found that monosaccharide rearrangement occurred during collision-induced dissociation (CID) of protonated glycopeptide ions. This effect resulted in pseudo Y1-glycopeptide fragment ions that indicated the presence of additional glycosylation sites on a single glycopeptide. CID oxonium ions and electron transfer dissociation, however, confirmed that just a single site was glycosylated, showing that glycan-to-peptide rearrangement can occur on glycopeptides and that this effect is influenced by the molecular nature of the glycan moiety. This effect was most pronounced with disaccharides. This study is the first report on O-linked flagellin glycosylation in a Selenomonas species, revealing that C9LY14 is one of the most heavily glycosylated flagellins described to date. This study contributes to our understanding of the largely under-investigated surface properties of oral bacteria. The data have been deposited to the ProteomeXchange with identifier PXD005859. Flagellar motility is one of the most extensively studied processes in prokaryotic microbiology. The bacterial flagellum, a complex multiprotein assembly, is best known as the locomotive organelle that allows microbes to actively move toward favorable environments by chemotaxis (1.Adler J. Chemotaxis in bacteria.Science. 1966; 153: 708-716Crossref PubMed Scopus (832) Google Scholar). In many pathogenic bacteria such as Escherichia coli, Vibrio cholerae, Helicobacter spp., Campylobacter spp., Pseudomonas aeruginosa, Borrelia burgdorferi, Treponema spp., and Salmonella spp. the flagellum represents an essential structure that is crucial for full pathogenesis (2.Josenhans C. Suerbaum S. The role of motility as a virulence factor in bacteria.Int. J. Med. 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Oral members of the bacterial genus Selenomonas have repeatedly been associated with periodontal disease and recent studies support the association of Selenomonas with the etiology of periodontitis, based on the detection of Selenomonas species at significant levels in subgingival biofilm samples of subjects with both chronic (17.Paster B.J. Boches S.K. Galvin J.L. Ericson R.E. Lau C.N. Levanos V.A. Sahasrabudhe A. Dewhirst F.E. Bacterial diversity in human subgingival plaque.J. Bacteriol. 2001; 183: 3770-3783Crossref PubMed Scopus (1482) Google Scholar) and aggressive (18.Faveri M. Mayer M.P. Feres M. de Figueiredo L.C. Dewhirst F.E. Paster B.J. Microbiological diversity of generalized aggressive periodontitis by 16S rRNA clonal analysis.Oral Microbiol. Immunol. 2008; 23: 112-118Crossref PubMed Scopus (122) Google Scholar) periodontitis. It appears that Selenomonas species contribute considerably to the structural organization of multispecies oral biofilms ("dental plaque"), which is the condition under which periodontitis develops (19.Drescher J. Schlafer S. Schaudinn C. Riep B. Neumann K. Friedmann A. Petrich A. Gobel U.B. Moter A. Molecular epidemiology and spatial distribution of Selenomonas spp. in subgingival biofilms.Eur. J. Oral Sci. 2010; 118: 466-474Crossref PubMed Scopus (32) Google Scholar). Periodontitis continues to be the most frequently occurring inflammatory disease world-wide; in its chronic form, it is the major cause of tooth loss and can also impact systemic health (20.Hajishengallis G. Periodontitis: from microbial immune subversion to systemic inflammation.Nat. Rev. Immunol. 2015; 15: 30-44Crossref PubMed Scopus (1313) Google Scholar), underlining the urgent need for therapeutic interference. However, although a group of distinct, nonmotile Gram-negative anaerobes (the "red complex") has been identified as keystone periodontal pathogens, the pathogenesis of periodontitis is still not fully understood (20.Hajishengallis G. Periodontitis: from microbial immune subversion to systemic inflammation.Nat. Rev. Immunol. 2015; 15: 30-44Crossref PubMed Scopus (1313) Google Scholar). Flagellation as present on S. sputigena cells might represent a hitherto unknown/unrecognized strategy of the bacterium to colonize its niche in the multispecies oral biofilm, thereby contributing to the establishment of the disease. To date there is essentially no knowledge available on flagellin glycosylation of oral Selenomonas spp. Unlike in mammalian glycoprotein synthesis, glycosylation pathways in bacterial species are highly diverse (21.Schaffer C. Messner P. Emerging facets of prokaryotic glycosylation.FEMS Microbiol. Rev. 2017; 41: 49-91Crossref PubMed Scopus (88) Google Scholar), making different orthogonal approaches a necessity to collect information on monosaccharide identity, glycan composition, and protein attachment. Mass spectrometry has always been a core technology for determining the primary structure of glycoprotein glycans. Glycopeptide product ion spectra provide a highly sensitive and selective opportunity to investigate glycosylation levels and sites of glycan attachment. Collision-induced dissociation (CID) 1The abbreviations used are: CID, collision-induced dissociation; AA, anthranilic acid; ACN, acetonitrile; BHI, Brain-Heart Infusion; CBB, Coomassie Brilliant Blue G-250; d-Gal, d-galactose; d-GalN, d-galactosamine; d-GalUA, d-galacturonic acid; d-Glc, d-glucose; d-GlcN, d-glucosamine; d-GlcUA, d-glucuronic acid; dHex, deoxyhexose; d-Man, d-mannose; d-Xyl, d-xylose; ETD, electron transfer dissociation; FLD, fluorescence detection; GdHCl, guanidinium hydrochloride; GlcNAc, N-acetylglucosamine; HexNAc, N-acetylhexosamine; IPTG, isopropyl β-d-1-thiogalactopyranoside; l-Ara, l-arabinose; LB, Luria-Bertani; l-Fuc, l-fucose; l-Rha, l-rhamnose; O-Me-Rha, O-methyl-rhamnose; PAS, periodic acid-Schiff; PBS, phosphate-buffered saline; PGC, porous graphitized carbon. 1The abbreviations used are: CID, collision-induced dissociation; AA, anthranilic acid; ACN, acetonitrile; BHI, Brain-Heart Infusion; CBB, Coomassie Brilliant Blue G-250; d-Gal, d-galactose; d-GalN, d-galactosamine; d-GalUA, d-galacturonic acid; d-Glc, d-glucose; d-GlcN, d-glucosamine; d-GlcUA, d-glucuronic acid; dHex, deoxyhexose; d-Man, d-mannose; d-Xyl, d-xylose; ETD, electron transfer dissociation; FLD, fluorescence detection; GdHCl, guanidinium hydrochloride; GlcNAc, N-acetylglucosamine; HexNAc, N-acetylhexosamine; IPTG, isopropyl β-d-1-thiogalactopyranoside; l-Ara, l-arabinose; LB, Luria-Bertani; l-Fuc, l-fucose; l-Rha, l-rhamnose; O-Me-Rha, O-methyl-rhamnose; PAS, periodic acid-Schiff; PBS, phosphate-buffered saline; PGC, porous graphitized carbon. of glycopeptides usually results in a prominent Y1-ion that facilitates identification of the peptide-linked monosaccharide (22.Ritchie M.A. Gill A.C. Deery M.J. Lilley K. Precursor ion scanning for detection and structural characterization of heterogeneous glycopeptide mixtures.J. Am. Soc. Mass Spectrom. 2002; 13: 1065-1077Crossref PubMed Scopus (76) Google Scholar). Such Y1-assigments are, however, increasingly difficult if more than one site of glycosylation is present on a single peptide or if gas phase monosaccharide rearrangements occur (23.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). Halim and Zauner independently reported the detection of small product ion signals in some O-glycopeptide product ion spectra acquired from human urinary and human fibrinogen glycopeptides that they assigned to a hexose rearrangement (24.Halim A. Nilsson J. Ruetschi U. Hesse C. Larson G. Human urinary glycoproteomics; attachment site specific analysis of N- and O-linked glycosylations by CID and ECD.Mol. Cell. Proteomics. 2012; 11 (M111 013649)Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 25.Zauner G. Hoffmann M. Rapp E. Koeleman C.A. Dragan I. Deelder A.M. Wuhrer M. Hensbergen P.J. Glycoproteomic analysis of human fibrinogen reveals novel regions of O-glycosylation.J. Proteome Res. 2012; 11: 5804-5814Crossref PubMed Scopus (38) Google Scholar). Because these pseudo Y1-glycopeptide signals were, if detected at all, just of very low intensity, they did not interfere with accurate assignment. Most of the monosaccharide rearrangements are reported for the analysis of protonated glycans, in particular when one or more fucose residues are present on the glycan (26.Wuhrer M. Deelder A.M. van der Burgt Y.E. Mass spectrometric glycan rearrangements.Mass Spectrom. 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In this study, we employed a variety of glycomic and glycoproteomic approaches to investigate the glycosylation of the S. sputigena flagellin protein (UniProt Accession C9LY14), demonstrating that C9LY14 is a heavily glycosylated protein exhibiting a surprising glyco-heterogeneity at multiple sites of O-glycosylation and carrying hitherto not described O-glycan structures. We also show that CID of O-glycan carrying glycopeptides induced a glycan structure dependent gas phase monosaccharide rearrangement resulting in the formation of strong pseudo Y1-ions. These indicated the presence of two sites of glycosylation on a single glycopeptide, whereas electron transfer dissociation (ETD) confirmed that a disaccharide was present on a single site of glycosylation only. Selenomonas sputigena ATCC 35185 was obtained from the Leibniz Institute DSMZ - German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany). Bacteria were grown anaerobically at 37 °C in 37 g/L Brain-Heart-Infusion (BHI) broth (Oxoid, Hampshire, UK), supplemented with 5 g/L yeast extract (Oxoid, Hampshire, UK), 0.5 g/L l-cysteine (Sigma), 5 μg/ml hemin (Sigma) and 2.0 μg/ml menadione (Sigma). For the preparation of BHI-agar plates (swarming plates), 0.5% w/v agar was added. Cells were harvested by centrifugation (5000 × g, 20 min, 4 °C), washed twice with phosphate-buffered saline (PBS; pH 7.5), and either used instantly or stored at −20 °C. Escherichia coli DH5α cells and E. coli BL21 (DE3) cells (both Life Technologies, Carlsbad, CA) were grown at 37 °C with shaking at 200 rpm in Luria-Bertani (LB) broth or on LB agar plates supplemented with 50 μg/ml kanamycin. Enrichment of native S. sputigena flagellin protein was achieved by differential centrifugation as described previously (27.Montie T.C. Craven R.C. Holder I.A. Flagellar preparations from Pseudomonas aeruginosa: isolation and characterization.Infect. Immun. 1982; 35: 281-288Crossref PubMed Google Scholar), with minor modifications. Briefly, bacterial cells from BHI-swarming plates were inoculated into 10 ml of BHI medium and incubated overnight at 37 °C. Five ml of this pre-culture were then transferred to 500 ml of half-strength BHI medium (diluted 1:1 with PBS) and incubated again overnight at 37 °C. Cells were harvested by centrifugation (5000 × g, 20 min) and resuspended in PBS (20 ml per 1 g of wet weight of cells). The suspension was blended for 2 min using a commercial blender to shear off flagella, followed by centrifugation (5000 × g, 30 min). The collected supernatant was centrifuged at 16,000 × g for 15 min and further processed by centrifugation at 40,000 × g for 3 h. All centrifugation steps were carried out at 4 °C. The remaining pellet was resuspended in 0.5 ml of Milli-Q (MQ) water and freeze-dried. The purity of the sample was analyzed by SDS-PAGE. SDS-PAGE was carried out on 12% slab gels according to Laemmli (28.Laemmli U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature. 1970; 227: 680-685Crossref PubMed Scopus (207002) Google Scholar). Protein bands were visualized using Coomassie Brilliant Blue G-250 (CBB; Serva, Heidelberg, Germany) and carbohydrates were stained with periodic acid-Schiff (PAS) reagent (29.Doerner K.C. White B.A. Detection of glycoproteins separated by nondenaturing polyacrylamide gel electrophoresis using the periodic acid-Schiff stain.Anal. Biochem. 1990; 187: 147-150Crossref PubMed Scopus (77) Google Scholar). For Western-immunoblotting, proteins were transferred onto a nitrocellulose membrane (Peqlab, Erlangen, Germany) and detection was performed at 800 nm on an Odyssey Infrared Imaging System (LI-COR, Lincoln, NE). For visualization of native flagellin C9LY14, a flagellin-specific antiserum was used in combination with IR Dye 800CW goat anti-mouse antibody (LI-COR). Detection of His6-tag on recombinant C9LY14 (C9LY14R) was done with a mouse anti-His6 antibody (Life Technologies) in combination with the IR Dye 800CW goat anti-mouse antibody (LI-COR). Restriction enzymes and ligase were purchased from Thermo Scientific (Vienna, Austria). Genomic DNA of S. sputigena ATCC 35185 was isolated as described previously (30.Cheng H.R. Jiang N. Extremely rapid extraction of DNA from bacteria and yeasts.Biotechnol. Lett. 2006; 28: 55-59Crossref PubMed Scopus (257) Google Scholar). Plasmid DNA was purified from transformed cells using the GeneJETTM Plasmid MiniPrep Kit (Thermo Scientific). PCR fragments were amplified by Phusion® High-Fidelity DNA Polymerase (Thermo Scientific) according to the manufacturer′s protocol. PCR fragments and digested plasmids were purified from agarose gels using the GeneJETTM Gel Extraction Kit (Thermo Scientific). Chemically competent E. coli DH5α cells and E. coli BL21 (DE3) cells were transformed according to the protocol provided by the manufacturer. Recombinant cells were analyzed by restriction mapping and confirmed by sequencing (Microsynth, Austria). Recombinant S. sputigena ATCC 35185 flagellin (C9LY14R) was expressed as C-terminally hexahistidine (His6)-tagged protein in E. coli BL21 (DE3). The coding sequence for C9LY14 was amplified from S. sputigena ATCC 35185 genomic DNA by PCR using the primers (Thermo Scientific) 002-fw-fla-NcoI (5′-CCGACACCATGGCATTGGTAGTTAAGAACAAC-3′) and 001-rev-fla-XhoI (5′-GACGACTCGAGCAGGCTGAGAACGCCGGAG-3′). The PCR product was digested with NcoI/XhoI and ligated into the pET28a(+) expression vector (Novagen, Wisconsin). Subsequently, pET28a_C9LY14R_His6 was transformed into E. coli BL21 (DE3) and freshly transformed cells were grown in LB medium at 37 °C with shaking at 200 rpm to an OD600 of ∼0.6. Protein expression was induced with a final concentration of 1 mm isopropyl β-d-1-thiogalactopyranoside (IPTG; Thermo Scientific) and cultures were grown for additional 3 h. His6-tagged C9LY14R for detection purposes was purified under denaturing conditions using Ni-NTA Agarose (Qiagen, Hilden, Germany) according to the manufacturer′s protocol. The purified protein was dialyzed against MQ water overnight at 4 °C. Escherichia coli BL21 (DE3) cells harboring the expression plasmid were harvested by centrifugation (5000 × g, 30 min, 4 °C) and resuspended in 50 mm sodium citrate buffer (pH 6.2) containing 0.1% Triton X-100. After addition of lysozyme (800 μg/ml; Sigma-Aldrich) and benzonase (50 U/ml; Sigma-Aldrich), cells were incubated for 30 min at 37 °C. Ultrasonication (Branson sonifier, duty cycle 50%; output 6) was used to further break open bacteria applying ten cycles of 10 pulses with 30-s breaks, each, and soluble protein was separated from the insoluble material by centrifugation (25,000 × g, 30 min, 4 °C), with both fractions containing C9LY14R. To increase the yield of C9LY14R, the remaining protein was extracted from the pellet with 50 mm sodium citrate buffer (pH 5.5) containing 5 m GdHCl, 20 mm imidazole and 0.5 m NaCl for 1.5 h at 4 °C and shaking at 200 rpm. The extract was centrifuged (45,000 × g, 1 h, 4 °C) and the supernatant subjected to membrane-filtering (0.45 μm pore size). The resulting protein extracts were combined and applied to a 1-ml HisTrap HP column (GE Healthcare, Little Chalfont, UK). The recombinant protein was recovered in elution buffer (50 mm sodium citrate buffer [pH 5.5], 5 m GdHCl, 1 m imidazole, 0.5 m NaCl) followed by dialysis against 50 mm sodium citrate buffer (pH 5.5). Immunization of mice and preparation of polyclonal antiserum against purified C9LY14R was done at EF-BIO s.r.o. (Bratislava, Slovakia). For glycoproteomic analyses, the preparation enriched in native S. sputigena flagellin was separated by SDS-PAGE and protein bands of interest were excised and destained (31.Kolarich 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 (157) Google Scholar). Any cysteine residues were reduced with 10 mm dithiothreitol (D0632; Sigma-Aldrich) for 1 h at 56 °C and carbamidomethylated by incubation in 55 mm iodoacetamide (I6125; Sigma-Aldrich) for 1 h at room temperature in the dark. Subsequently, the protein was in-gel digested at 37 °C with trypsin (11047841001; Roche, Basel, Switzerland) or chymotrypsin (11418467001; Roche), respectively, in 25 mm ammonium bicarbonate buffer using an enzyme-to-protein ratio of 1:50 (w/w). Tryptic digests were carried out overnight, whereas chymotrypsin digests were terminated after 4 h. Resulting (glyco)peptides were extracted from the gel pieces and dried in a centrifugal evaporator (in vacuo). For reversed-phase nanoLC-ESI-MSMS analyses, the samples were resolubilized in 40 μl of 0.1% formic acid (94318; Sigma-Aldrich) from which 1-μl aliquots were injected per analysis. Glycoproteomic data has been acquired for three flagellin preparations that were performed on three independently grown S. sputigena cultures. A technical triplicate analysis was performed from a single batch for the in E. coli-expressed recombinant flagellin protein. O-glycans were released from tryptic glycopeptides by reductive β-elimination. Dried glycopeptides were incubated with 50 μl of 0.5 m sodium borohydride (71321; Sigma-Aldrich) in 50 mm potassium hydroxide (221473; Sigma-Aldrich) for 16 h at 50 °C. The released O-glycans were desalted over an AG 50W-X8 resin (142–1451; Bio-Rad Laboratories, Hercules, CA) and purified by solid-phase extraction with porous graphitized carbon (PGC; 210101, Grace Bio-Labs, Oregon) (32.Jensen P.H. Karlsson N.G. Kolarich D. Packer N.H. Structural analysis of N- and O-glycans released from glycoproteins.Nat. Protoc. 2012; 7: 1299-1310Crossref PubMed Scopus (284) Google Scholar). For glycomic analyses by PGC nanoLC-ESI-MSMS, the dried O-glycans were resolubilized in 15 μl of 10 mm ammonium bicarbonate of which 5 μl were injected per analysis. Because of limitations in sample material, glycomic data has been acquired for a single S. sputigena flagellin preparation. Peptides and glycopeptides were analyzed by reversed-phase nanoLC-ESI-MSMS on a Dionex Ultimate 3000 UHPLC online coupled to a Bruker amaZon Speed ETD ion trap mass spectrometer in positive ion mode. Reversed phase chromatography was performed on PepMap C18 columns (precolumn: Thermo Fisher, PepMap100, 100 μm [ID] × 2 cm, C18, 5 μm particle size, 100 Å pore size, P/N 164564; analytical column: Thermo Fisher, Acclaim PepMap RSLC, 75 μm [ID] × 15 cm, C18, 2 μm particle size, 100 Å pore size, P/N 164534) using a linear gradient of 0.1% formic acid (solvent A) and 90% acetonitrile (ACN) containing 0.1% formic acid (solvent B) at a constant flow rate of 400 nl/min at 45 °C. The samples were loaded onto the trap column in solvent A. The analytical gradient started at 5% of solvent B and gradually increased to 45% over 30 min. The columns were flushed with 90% solvent B for 10 min after each sample before re-equilibrating to starting conditions. The ion trap was set to scan from m/z 400 to 1600 with an SPS Target Mass of m/z 1350 using the instrument's Enhanced Resolution mode. The ICC target was set to 200,000 with a maximum accumulation time of 50 ms. The three most intense signals of each MS scan were selected for CID and the resulting fragments were recorded from m/z 100 to 2000 in the UltraScan mode. Besides CID, electron transfer dissociation (ETD) was employed for (glyco)peptide fragmentation in separate analyses. The ETD reagent (flouranthene) ICC Target was set to 500,000 with a maximum accumulation time of 10 ms. The ETD reaction time was set to 100 ms. The glycopeptide signal intensities were further enhanced using a Bruker CaptiveSpray NanoBooster™ ionization source using nitrogen as dry gas (3 L/min at 150 °C) and ACN as dopant solvent. An Active Exclusion setting (exclude after three spectra, release after 0.5 min) was employed in the data-dependent acquisition (DDA) method. The acquired MSMS data was analyzed using Bruker Data
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