Diversity in the Protein N-Glycosylation Pathways Within the Campylobacter Genus^*

Plasmids, Bacterial Strains, and Growth Conditions

All bacterial strains and plasmids used in this study are listed in Table I. Campylobacter strains were grown on Mueller Hinton or Brain Heart Infusion supplemented with 5% horse blood under microaerobic (85% N₂, 10% CO₂, and 5% O₂) conditions. Alternatively, anaerobic strains were grown for 18–72 h at 37 °C in the following medium: 30 g/l Tryptic Soy Broth, 2 g/l yeast extract, 3.0 g/l Bacto-Peptone, 2.0 g/l NaCl, 3.0 g/l sodium formate, 4.0 g/l formic acid, 4.0 g/l asparagine monohydrate, 1.0 g/l cysteine hydrochloride, pH 7.8; supplemented with 5% (v/v) horse blood. For large scale anaerobic cultures, bacteria were grown in anaerobic medium (without horse blood) at 37 °C in ventilated 1 liter bottles in anaerobic jars using the AnaeroGen gas pack system (Oxoid). E. coli strains were routinely grown in 2× YT medium at 37 °C. Growth media were supplemented with chloramphenicol (Cm, 25 μg/ml) and/or kanamycin (Kan, 30 μg/ml), where appropriate.

Phylogenetic Analysis

The AtpA (ATP synthase α subunit, housekeeping gene) protein sequence was used as the phylogenetic marker for trees constructed with the neighbor-joining algorithm of the MEGA3.1 software package (20) using a multiple alignment parameters gap opening penalty of 8, a gap extension penalty of 2, and the PAM protein weight matrix. To identify putative pgl genes, the predicted proteomes of 29 Campylobacter genomes, representing all validly described taxa, were compared with the predicted proteome of C. jejuni strain NCTC 11168 using BLASTP. Proteins demonstrating >35% amino acid identity to Pgl proteins from 11168 were scored as positive matches. Putative gene products within the 29 proposed pgl loci were identified by BLASTP comparison to proteins present within the NCBI nonredundant (nr) database. Annotation of the pgl loci also used the Sanger Centre Pfam protein families database (v 26.0; Nov. 2011) (21) and the CAZy database (22) to identify additional putative glycosyltransferases.

fOS Purification and NMR Analysis

Campylobacter cells from 6 liter cultures were harvested (10 min, 10,000 × g, 4 °C) and digested with proteinase K at pH 8 at 37 °C for 48 h. Digests were separated on a Sephadex G-15 column (1.5 × 60 cm). Fractions were dried, resuspended in D₂O and analyzed by ¹H NMR. Fractions containing fOS were separated by anion exchange chromatography on a Hitrap Q column (5 ml size, Amersham Biosciences). Glycans were eluted with a linear gradient of NaCl (0–1 M, 1 h). Desalting was performed on Sephadex G15 prior to glycan analysis on a Varian INOVA 500 MHz (¹H) NMR spectrometer with a 3 mm gradient probe at 25 °C with acetone as the internal reference (2.225 ppm for ¹H, 31.45 ppm for ¹³C) using standard pulse sequences: DQCOSY, TOCSY (mixing time 120 ms), ROESY (mixing time 500 ms), HSQC and HMBC (100 ms long range transfer delay). AQ time was kept at 0.8–1 s for H-H correlations and 0.25 s for HSQC, 256 increments was acquired for t1. Sugars and sugar linkages were identified by characteristic coupling constants and chemical shift patterns. For fOS of C. concisus, the absolute configuration of the glucolactilic acid carboxyethyl group was determined as described (23).

HPAEC-PAD

Cff fOS were separated by high performance anion exchange chromatography with pulsed amperometric detection (HPAEC/PAD) using a semipreparative CarboPac® PA 100 (9 × 250 mm equipped with a Guard Column: 3 × 50 mm) and a fraction collector (DIONEX UltiMate 3000) under the following conditions: flow rate: 0.5 ml/min; eluent system: 50 mm sodium acetate in 100 mm sodium hydroxide; detection mode: pulsed amperometry, quadruple waveform, Au electrode; AgCl reference electrode; and the ambient column temperature was set to ∼30 °C. Elution fractions containing the single fOS forms were neutralized with equimolar amounts of 0.2 m HCl and 1/10 of each elution fraction was applied to an analytical column CarboPac® PA200 (3 × 250 mm CarboPac PA100 equipped with a Guard Column: 3 × 50 mm) under the same conditions to confirm proper separation of individual fOS forms. Individual fOS-containing fractions were combined, lyophilized and stored at −20 °C until further use.

fOS Analysis Using Mass Spectrometry

fOS were identified and analyzed as described (8, 24), with the difference that precursor ion scanning was carried out for the detection of the diagnostic oxonium ion fragments at m/z 204 (for HexNAc) and m/z 407 (HexNAc)₂.

Preparation of Whole-cell Lysates and Immunoblotting

Whole-cell lysates were prepared as described (8). Immunoblotting using R1, hR6 (25), GRPII-1 (anti-Cff-minor), GRPII-2 (anti-Cff-major), or alkaline phosphatase (AP)-conjugated wheat germ agglutinin (WGA) (Vector Laboratories (Canada), Inc.) was performed as described (24) with dilutions of 1:10,000 for each primary antiserum, 1:2000 for WGA-AP and 1:2000 for the secondary AP-labeled antibody (Santa Cruz Biotechnology, Santa Cruz, CA).

Generation of fOS-BSA Glycoconjugates

Individual Cff fOS preparations were conjugated to BSA by reductive amination as described (26). Formation of BSA-glycoconjugates was confirmed by Western blotting with alkaline phosphatase (AP)-conjugated WGA.

Generation of Group I and Group II C. fetus Subgroup Glycan-specific Antibodies

New Zealand White Rabbits were immunized with individual group II C. fetus subgroup fOS-BSA conjugates using a 10 week immunization protocol (approved Animal Care Committee protocol No. 717). Blood sera were analyzed for the production of pgl glycan-specific antibodies by Western blotting against whole cell lysates from various Campylobacter species. The antiserum against HexNAc-[Hex]-HexNAc₃-diNAcBac-BSA was named GRPII-1, the antiserum against HexNAc-[HexNAc]-HexNAc₃-diNAcBac-BSA was named GRPII-2. To generate group I-N-glycan specific (R1) antiserum, rabbits were immunized with formaldehyde-fixed (according to (27)) cells of E. coli K12 wzy::Kan (pACYC(pgl_mut) expressing the C. jejuni N-glycan on their surface (17).

Digestion of Whole-cell Lysates for ZIC-HILIC Enrichment

One mg of proteins, determined using the Qubit^TM kit (Invitrogen, Carlsbad CA), was resuspended in 6 m urea, 2 m thiourea, and 40 mm NH₄HCO_3. The solubilized sample was then reduced for 1 h with 10 mm dithiothreitol, subsequently alkylated using 20 mm iodoacetamide for 1 h in the dark and was quenched using 10 mm dithiothreitol. Then, 1/200 (w/w) of endoproteinase Lys-C (Sigma-Aldrich) was added and digestion was allowed to proceed for 4 h at 25 °C. Samples were diluted 1:4 with 100 mm NH₄HCO₃ and digested with 1/50 (w/w) of porcine sequencing grade trypsin (Promega, Madison, WI) for 18 h at 25 °C. Samples were then dialyzed against ultra-pure water overnight using a Mini Dialysis Kit with a molecular mass cut off of 1000 Da (GE Healthcare) and lyophilized.

Enrichment of Glycopeptides by Zwitterionic Hydrophilic Interaction Chromatography (ZIC-HILIC)

ZIC-HILIC enrichment was performed according to (4) with minor modifications. Micro-columns composed of 10 μm ZIC-HILIC resin (Sequant, Umeå, Sweden) packed into p10 tips containing a 1 mm² excised C₈ Empore™ disc (Sigma) were packed to a bed length of 0.5 cm. Before use, the columns were washed with ultra-pure water, followed by 95% acetonitrile (ACN) and then equilibrated with 80% ACN and 5% formic acid (FA). Samples were resuspended in 80% ACN, 5% FA and insoluble material removed by centrifugation at 20,000 × g for 5 min at 4 °C. Samples were adjusted to a concentration of 2 μg/μl and 100 μg of peptide material loaded onto a column and washed with 10 load volumes of 80% ACN, 5% FA. Peptides were eluted with three load volumes of ultra-pure water into low-bind tubes and concentrated using vacuum centrifugation. Triplicate enrichments were generated for each glycopeptide analysis.

Identification of Glycopeptides Using Reversed Phase LC- MS and HCD MS/MS

ZIC-HILIC enriched glycopeptide samples were resuspended in 0.1% FA and loaded onto an Acclaim PepMap 100 μm C18 Nano-Trap Column (Dionex Corporation, Sunnyvale, CA) for 10 min using a UltiMate 3000 intelligent LC system (Dionex Corporation). Peptides were eluted and separated using a 20 cm, 100 μm id, ReproSil - Pur C₁₈ AQ 3 μm column using a 120 min gradient in which the proportion of phase A (0.1% FA) and phase B (0.1% FA, 80% ACN) were altered from 100% phase A to 40% phase B over 120 min at 200 nL/min in an LTQ-Orbitrap Velos mass spectrometer (Thermo Scientific, San Jose, CA). The LTQ-Orbitrap Velos was operated using Xcalibur v2.2 with a capillary temperature of 275 °C in a data-dependent mode automatically switching between MS and MS/MS with CID followed by HCD. For each MS scan, the three most abundant precursor ions were selected for fragmentation with CID (normalized collision energy 35, activation time 30 ms) followed by HCD (normalized collision energy 45, activation time 30 ms) to obtain MS/MS spectra. MS resolution was set to 60,000 with an ACG of 1e⁶, maximum fill time of 500 ms and a mass window of 600 to 2000 m/z. CID fragmentation was carried out with an ACG of 2e⁴, maximum fill time of 100 ms whereas HCD fragmentation was carried out with an ACG of 2e⁵, maximum fill time of 500 ms, resolution of 7500 and mass window 170 to 2000 m/z.

Glycopeptide Data Processing

The .raw files were then processed within Proteome Discover version 1.0 Build 43 (Thermo Scientific) to generate mgf files and searched using Sequest against the specific strain FASTA database (obtained from NCBI on 08/06/2011). Scan events that did not result in peptide identification from Sequest searches were exported to Excel (Microsoft, Redmond, WA). To identify possible glycopeptides within this list, a new feature within the MS² module of GPMAW 8.2 called “mgf graph” was used, which identified all scan events within the generated mgfs containing the diagnostic oxonium 204.086 m/z ion. Using Excel, all scan events that were not matched by Sequest and contained a predicted marker of glycosylation were identified. These events were manually inspected and identified as possible glycopeptides based on the presence of the glycan fragment within the CID scan. To facilitate glycopeptide assignments from HCD scans, the ions below the mass of the predicted deglycosylated peptides were extracted with Xcalibur v2.2 using the Spectrum list function. Ions with a deconvoluted mass above the deglycosylated peptide and ions corresponding to known carbohydrate oxoniums were removed in a similar approach to post-spectral processing of ETD data (28, 29). MASCOT v2.2 searches were conducted via the Australasian Proteomics Computational Facility (www.apcf.edu.au) of the proteobacteria taxonomy of the LugwigNR database (database release LugwigNR_Q410, composed of 13112897 entries available via ftp://ftp.ch.embnet.org/pub/databases/nr_prot/). Searches were carried out with a parent ion mass accuracy of 20 ppm and a product ion accuracy of 0.02 Da with no protease specificity as well as the fixed modification carbamidomethyl (C) and variable modifications, oxidation (M) and deamidation (N). The instrument setting of MALDI-QIT-TOF was chosen because of previous studies showing quadrupole-like fragmentation within HCD spectra (30) (generating a, b, and y ions) and our observation of internal cleavage products that are all included in this setting. All spectra were searched with the decoy option enabled and no matches to this database were detected (FDR 0%). To further validate glycopeptide matches, all spectra were manually annotated to ensure all major peaks were match providing further confidence of identity and localization. All annotated spectra are provided in supplemental MS-Figures.

Campylobacters Divide into Two Major Groups Based on Phylogeny and Antigenicity

All Campylobacter species, except C. canadensis, possess a complete repertoire of pgl genes (Fig. 1). Phylogenetic comparisons showed that campylobacters divide into two major groups that comprise the thermotolerant (group I) and the nonthermotolerant species (group II) with C. lari and related species forming a subgroup (the C. lari subgroup), within group I (Fig. 1). When whole cell lysates of all 29 Campylobacter pgl-containing species were screened with C. jejuni N-glycan specific antisera, all group I organisms were recognized (Figs. 2A, 2B). No reactivity against proteins from the group II species was observed suggesting that these organisms are either incapable of synthesizing N-glycans or they produce different oligosaccharide structures compared with C. jejuni.

The Characterization of Group I Campylobacter fOS Structures

Because the C. jejuni fOS to N-glycan ratio is 10:1 under standard laboratory growth conditions (9), we screened species from each group for fOS by mass spectrometry using precursor ion scanning. In group I organisms, except the C. lari subgroup, a mass of m/z 1425.0 strongly suggested the presence of a heptasaccharide structure similar to C. jejuni (supplemental Fig. S1) (9). For fOS isolated from the C. lari subgroup, masses of m/z 1263.0, m/z 1244.6, and 1343.0 were detected (supplemental Fig. S1). In C. lari subsp. lari, MS² analysis of m/z 1263.0 and 1245.6 indicated a linear [HexNAc]₅-diNAcBac oligosaccharide composition and its nonhydrated form (Figs. 3A, 3B) whereas m/z 1344.0 correlated to the hexasaccharide with phosphate attached, ([HexNAc]₅-diNAcBac-P; Fig. 3C). The presence of phosphate was confirmed by the neutral loss of phosphoric acid (∼98 m/z) from the 1344.0 m/z structure (31).

Because multiple glycoforms were identified from the C. lari subsp. lari fOS analysis, we used NMR to determine the phosphate position on the fOS and investigate the authenticity of the nonphosphorylated form (Fig. 6, supplemental Table S2). For the C. lari subsp. lari fOS, the NMR H-1 signal of diNAcBac showed correlation to a phosphate signal at 1 ppm and had an additional coupling of 7 Hz because of phosphorylation. The C-1 signal was at 95.2 and no beta anomer was present. Therefore the phosphorylation was at the O-1 of diNAcBac and the linear [GalNAc]₅-diNAcBac-P structure was confirmed for this species with no evidence of dephosphorylated glycoforms detected. As a control, structural analysis of fOS from C. jejuni (Fig. 6, supplemental Table S3) confirmed the previously established N-linked glycan structure is identical to the fOS glycan.

The Characterization of Group II Campylobacter fOS Structures

As representatives of the group II species, we analyzed Cff, Cfv, C. hyointestinalis subsp. hyointestinalis (Chyo) and C. hyointestinalis subsp. lawsonii (further referred to as the C. fetus subgroup). Precursor ion scanning for the diagnostic oxonium ion m/z 204 (for HexNAc) identified two parent ions with m/z 1263.0 and m/z 1222.0 (supplemental Fig. S1). MS² analyses of these ions for Cff identified the monosaccharide compositions and sequence of both fOS species as HexNAc-[Hex]-HexNAc₃-diNAcBac and HexNAc-[HexNAc]-HexNAc₃-diNAcBac (Figs. 3D, 3E). NMR analysis of the fOS of Cff, Cfv and Chyo confirmed that these species produce two oligosaccharides, as suggested by MS analysis (Fig. 6, supplemental Table S1). Within these glycans the three sugars at the reducing end were identical to those of C. jejuni whereas the three sugars at the nonreducing end were: β-GlcNAc-3-[α-GlcNAc-6]-α-GlcNAc-4 and β-Glc-3-[α-GlcNAc-6]-α-GlcNAc-4.

The presence of two oligosaccharide variants in the C. fetus subgroup raised the question about their expression and antigenicity. Quantitative and preparative HPAEC-PAD analyses on purified C. fetus subgroup II glycans (Fig. 4) not only allowed the separation of the two Cff-fOS species, but also demonstrated that the ratio of peak 1 (major)/peak 2 (minor) group II fOS was ∼3.6:1 (Fig. 4). Immunofluorescence staining of intact cells of Cff, Cfv and Chyo with GRPII-1 and GRPII-2 antisera individually raised in rabbits against each of the two structures, showed that both glycans are antigenic and expressed on the bacterial cell surface (Fig. 4F). In contrast, the GRPII antisera did not react with C. jejuni (group I) cells (Fig. 4F). Similarly, group II species could not be labeled with group I antiserum whereas C. jejuni cells showed strong reactivity.

To confirm that these glycoforms are indeed N-linked to protein, Cff whole cell lysates were subjected to ZIC-HILIC glycopeptide enrichment and MS-MS analysis. A total of 65 unique glycopeptides were identified within this species (supplemental Table S7) with both glycans detected on protein substrates. Forty-six peptides contained the HexNAc-[HexNAc]-HexNAc₃-diNAcBac glycan whereas 19 contained the HexNAc-[Hex]-HexNAc₃-diNAcBac glycan with 14 glycosylation sites confidently assigned independent of sequon alone (supplemental Table S9) and included examples of both glycans occupying the same glycosylation site. The identification of glycopeptides containing both glycans, which co-eluted, enabled the comparison of the ratio of the HexNAc-[HexNAc]-HexNAc₃-diNAcBac and HexNAc-[Hex]-HexNAc₃-diNAcBac glycoforms of the 19 glycopeptide pairs detected (supplemental Table S8). An example of this is the glycopeptide ¹³⁵DTNETELSAQK¹⁴⁵ of the putative protein A0RNV0_CAMFF, which demonstrates the ratio of ∼3.5/1 for the two glycoforms (Fig. 5) that is representative for the average, ∼3.7/1, observed for all confirmed glycopeptide pairs (supplemental Table S8). This ratio is in good agreement with the observed fOS ratio.

Group II Campylobacter Species Show Extensive Diversity and Split into Eight Subgroups

The generation of C. fetus specific glycans antibodies provided a rapid screening tool to investigate the attachment of similar glycans to protein substrates. Whole-cell lysates of all 29 Campylobacter species were probed with GRPII-1/GRPII-2 N-glycan specific antisera (Figs. 2C, 2D). Similar to the immunofluorescence results, proteins of Group I species did not react, whereas proteins from C. fetus sp. and C. hyointestinalis sp. strongly reacted with GRPII sera. The GRPII sera showed variable reactivity with other group II Campylobacter species, which ranged from moderate with C. gracilis proteins, weak with C. sputorum bv. sputorum and C. lanienae, and no reactivity against C. hominis, C. rectus, C. showae, C. mucosalis, C. concisus, C. curvus, C. ureolyticus, C. sputorum bv. fecalis and C. sputorum bv. paraureolyticus (Figs. 2C, 2D). Interestingly Campylobacter species with a closer phylogenetic relationship demonstrated similar patterns of reactivity toward GRPII-1/GRPII-2 antisera suggesting that group II may contain subgroups that differ in their glycan structures. Because of the observed variations in antibody reactivity, we analyzed the fOS composition of the remaining group II strains by MS and MS². In the C. sputorum subgroup (including C. lanienae and C. sputorum bvs. sputorum, paraureolyticus, and fecalis), precursor ion scanning identified masses at m/z 1384.4 and 1425.0 (supplemental Fig. S1) corresponding to Hex-HexNAc-[Hex]-HexNAc₃-diNAcBac and HexNAc₂-[Hex]-HexNAc₃-diNAcBac, respectively (Figs. 3F, 3G). The ion at m/z 528.2 (Fig. 3G) may represent [Hex]₂-HexNAc and thus suggests the branch to be terminal. For C. ureolyticus and C. concisus, m/z 1456.0 (supplemental Fig. S1) represents 234-HexNAc-[Hex]-HexNAc-HexNAc₂-diNAcBac (Fig. 3H) whereas in C. showae and C. rectus, precursor ion scans identified a parent ion at m/z 1560.0 (supplemental Fig. S1). MS² strongly suggested the composition [Hex]₃, [HexNAc]₃, [217]₁ and [diNAcBac]₁, but the full oligosaccharide sequence could not be solved (Fig. 3I). Similarly, in C. hominis, a parent ion at m/z 1497.5 (supplemental Fig. S1) most likely reflects a sugar composition of [234]₁ [HexNAc]₅ and [diNAcBac]₁ (Fig. 3J) whereas for C. curvus and C. mucosalis, identical masses of m/z 1439.6 (supplemental Fig. S1) represent HexNAc₂-[Hex]-HexNAc-217-HexNAc-diNAcBac (Fig. 3K), For C. gracilis, a parent ion mass at m/z 1417.4 (supplemental Fig. S1) represents a hexasaccharide with the sequence 123-HexNAc-[234]-HexNAc₃-diNAcBac (Fig. 3L). The fragment ions with m/z 626.8, 487.1 and 327.7 were indicative of a PEtN residue linked to an acetylated amino sugar (HexNAc). Interestingly, fragmentation of this PEtN containing glycan was dominated by B-ions compared with the typically Y-ion series dominant spectra seen for the other fOS glycans.

The Characterization of Group II Subgroups by the Identification of fOS Structures

Because fOS analysis identified the presence of unusual monosaccharides (m/z 234 and m/z 217) and we detected differences in glycoprotein reactivity with GRPII-1/GRPII-2 antisera within the C. sputorum subgroup (although MS² suggests similar fOS sequences), we solved the representative fOS structures of each subgroup by NMR. Within C. concisus, the 234 Da component is glucolactilic acid (Glc4RLac) and the structure of the fOS was assigned to be Glc4RLac-6-α-GalNAc-4-[α-Glc-6]-α-GalNAc-4-α-GalNAc-4-α-GalNAc-3-α,β-diNAcBac (Fig. 6, supplemental Table S6). The position of the glucolactilic acid was determined based on strong NOEs between H-2 of the lactilic acid and H-4 of the glucose and on the low field chemical shift of the glucose C-4 (78.5 ppm) that was caused by alkylation of the Glc O-4 by C-2 of lactilic acid (data not shown). For C. sputorum bv. sputorum the structure was determined to be β-Glc-4-β-GlcNAc-3-[α-GlcNAc-6]-α-GlcNAc-4-α-GalNAc-4-α-GalNAc-3-α,β-diNAcBac (Fig. 6, supplemental Table S4), whereas the structure for C. sputorum bv. fecalis is β-Glc-4-β-GlcNAc-3-[α-GalNAc-6]-α-GlcNAc-4-α-GalNAc-4-α-GalNAc-3-α,β-diNAcBac (Fig. 6, supplemental Table S5). Interestingly, these fOS structures only differ by the configuration of the HexNAc branch. Although multiple attempts were made to analyze fOS from all representative subgroups, we were not able to obtain the amount of fOS that is required for NMR to elucidate the glycan structure of the remaining Campylobacter species.

Characterization of Group II Glycans by Glycopeptide Enrichment

Because we could not fully elucidate the fOS structure of certain Campylobacter group II species such as C. rectus and C. hominis, by MS or NMR, we employed ZIC-HILIC glycopeptide enrichment to further characterize these glycans and their composition. Enrichment of C. hominis glycopeptides confirmed that the fOS composition of [234]₁, [HexNAc]₅, [diNAcBac]₁ was indeed found on protein substrates such as the preprotein translocase, SecG subunit ⁹³EDQNKSEPFVPLVPDAK¹⁰⁹ and shown to be: HexNAc₂-[234]-HexNAc₃-diNAcBac (Figs. 7A, 7D). Additional variations in the glycopeptides were detected that varied in the composition of the two terminal sugars. HCD and CID fragmentation patterns clearly showed the presence of two variants that contain a unique mass of m/z 245 (accurate mass analysis exhibited MH⁺ = 246.09506): 245-HexNAc-[234]-HexNAc₃-diNAcBac and 245–245-[234]-HexNAc₃-diNAcBac (Figs. 7B, 7C). Within the glycopeptide analysis, isobaric glycoforms of the 4271.61 Da glycan were noted (Fig. 8A). This mass was found to correspond to two variants; 245-HexNAc-[234]-HexNAc₃-diNAcBac and HexNAc-245-[234]-HexNAc₃-diNAcBac (Figs. 8B, 8C), which differ in the position of the 245.09 carbohydrate moiety but are attached to the same protein substrate (foldase protein PrsA, ²⁶³AANVQFTDTNATK²⁶⁵ Fig. 8D). In total 18 unique glycopeptides corresponding to 10 unique peptide sequences and five localized glycosylation sites were identified within C. hominis (supplemental Table S10) with this being the first documented case of isobaric N-linked glycans within the Campylobacter genus.

Analysis of C. rectus glycopeptides confirmed the fOS mass of 1560.0 Da and also enabled the complete assignment of the oligosaccharide as Hex-HexNAc-[Hex-Hex]-HexNAc-217-HexNAc-diNAcBac (Fig. 9A). Within the generated HCD spectra, the unusual 217 Da carbohydrate moiety lead to the formation of an oxonium ion of mass MH⁺ = 218.06398 that strongly suggested an elemental composition of C6H10O5N4 (-2.710ppm) with the presence of the oxonium ion being diagnostic for C. rectus glycopeptides. A total of 25 unique glycopeptides were identified enabling the localization of 11 glycosylation sites across multiple proteins (supplemental Table S11) such as the M23 peptidase domain protein, ⁴³⁸KAEENLTK⁴⁴⁵ (Fig. 9B). The C. rectus N-glycan represents the first example of an octasaccharide within the Campylobacter genus.

Confirmation of a Noncarbohydrate Moiety in the C. gracilis Glycans by Glycopeptide Enrichment

Because our initial analysis of C. gracilis fOS lead to the identification of a glycan composition of 123-HexNAc-[234]-HexNAc₃-diNAcBac with the identity of the 123 Da moiety being unassigned, we further characterized this moiety and verified the attachment of this unusual glycan to protein substrates by ZIC-HILIC glycopeptide enrichment. Multiple glycoforms were identified in C. gracilis that were characterized by the presence or absence of the noncarbohydrate modification PEtN. In total eight glycopeptides from seven glycoproteins were identified enabling the localization of four glycosylation sites (supplemental Table S12). Within this dataset, two proteins and three glycopeptides were demonstrated to contain PEtN on the glycan (supplemental Table S12). Examples of the identified C. gracilis glycopeptides were the PEtN-HexNAc-[234]-HexNAc₃-diNAcBac glycopeptide, ⁸⁴VKIEANATK⁹² of preprotein translocase SecG subunit (Fig. 9C, D) and the HexNAc-[234]-HexNAc₃-diNAcBac glycopeptide, ¹¹⁷TGADLNGTLAEQR¹²⁹ of the periplasmic nitrate reductase (Figs. 9E, 9F). Within this dataset, the use of CID and HCD fragmentation led to the generation of internal PEtN fragments, including the loss of the diagnostic ethanolamine group at 43.04 Da, which aided in the assignment of this noncarbohydrate modification.

The N-linked Glycan of C. lari subsp. lari Differs from Its fOS Counterpart

To determine whether the N-glycan of C. lari, like its fOS counterpart, is modified with a phosphate residue at the reducing end, ZIC-HILIC enrichment and a combination of MS HCD and ETD fragmentation was used to identify glycopeptides in C. lari subsp. lari (supplemental Table S13). Within the identified glycopeptides, such as ⁵⁸AAVDANASGSEK⁶⁹ from ubiquinol cytochrome c oxidoreductase, only [HexNAc]₅-diNAcBac glycans were detected (Figs. 9G, 9H) and there was no evidence of phosphate-containing N-glycan suggesting that the C. lari subsp. lari N-linked glycan is [HexNAc]₅-diNAcBac.