Simultaneous glycan-peptide characterization using hydrophilic interaction chromatography and parallel fragmentation by CID, higher energy collisional dissociation, and electron transfer dissociation MS applied to the N-linked glycoproteome of Campylobacter jejuni

Abstract

Campylobacter jejuni is a gastrointestinal pathogen that is able to modify membrane and periplasmic proteins by the N-linked addition of a 7-residue glycan at the strict attachment motif (D/E)XNX(S/T). Strategies for a comprehensive analysis of the targets of glycosylation, however, are hampered by the resistance of the glycan-peptide bond to enzymatic digestion or β-elimination and have previously concentrated on soluble glycoproteins compatible with lectin affinity and gel-based approaches. We developed strategies for enriching C. jejuni HB93-13 glycopeptides using zwitterionic hydrophilic interaction chromatography and examined novel fragmentation, including collision-induced dissociation (CID) and higher energy collisional (C-trap) dissociation (HCD) as well as CID/electron transfer dissociation (ETD) mass spectrometry. CID/HCD enabled the identification of glycan structure and peptide backbone, allowing glycopeptide identification, whereas CID/ETD enabled the elucidation of glycosylation sites by maintaining the glycan-peptide linkage. A total of 130 glycopeptides, representing 75 glycosylation sites, were identified from LC-MS/MS using zwitterionic hydrophilic interaction chromatography coupled to CID/HCD and CID/ETD. CID/HCD provided the majority of the identifications (73 sites) compared with ETD (26 sites). We also examined soluble glycoproteins by soybean agglutinin affinity and two-dimensional electrophoresis and identified a further six glycosylation sites. This study more than doubles the number of confirmed N-linked glycosylation sites in C. jejuni and is the first to utilize HCD fragmentation for glycopeptide identification with intact glycan. We also show that hydrophobic integral membrane proteins are significant targets of glycosylation in this organism. Our data demonstrate that peptide-centric approaches coupled to novel mass spectrometric fragmentation techniques may be suitable for application to eukaryotic glycoproteins for simultaneous elucidation of glycan structures and peptide sequence.

Fig. 1.

ZIC-HILIC glycopeptide enrichment of C. jejuni glycoprotein PEB3 (NCBI database accession number YP_002343730) digested with trypsin. A, MALDI-MS of trypsin-digested PEB3; labeled peaks correspond to non-glycosylated PEB3 peptides. B, MALDI-MS of ZIC-HILIC enriched PEB3 tryptic peptides; the dominant 2114.1813 m/z peak corresponds to the known glycopeptide ⁸⁸DFNVSK⁹³ with addition of the C. jejuni glycan (1405.560 Da). Peaks labeled with * correspond to non-glycosylated tryptic peptides. Na⁺, sodium adduct of 2114.18-Da glycopeptide. The chemical structure of the C. jejuni heptasaccharide is shown in Weerapana and Imperiali (47).

Fig. 2.

Comparison of CID-, HCD-, and ETD-MS for fragmentation of C. jejuni PEB3 glycopeptide 2114.1813 m/z. A, CID-MS; the MS/MS spectrum is dominated by fragment ions generated from the glycan (depicted according to the nomenclature of Domon and Costello (42)). B, HCD-MS; eight of 10 peptide-related b and y ions were detected with a mass accuracy of 0.02 Da. The diagnostic 204.086 m/z GalNAc ion and complete peptide (709.35 m/z) attached to the linking bacterial sugar bacillosamine (937.46 m/z) were also detected. C, ETD-MS; exclusive c and z fragment ions (four peptide-related fragment ions could be assigned). ETD is dominated by charge-reduced species and multiple ions corresponding to neutral loss species (denoted with &).

Fig. 3.

Comparison of CID/HCD and CID/ETD glycopeptide identifications from C. jejuni HB93-13. A total of 75 sites were identified.

Fig. 4.

Novel C. jejuni glycosylation sites identified using HCD- and ETD-based approaches. HCD-MS (A) and ETD-MS (B) of the glycopeptide ⁷⁰NCGDFNK⁷⁶ from putative lipoprotein Cj0089c are shown. C, HCD-MS of the glycopeptide ²⁸²DNNLSLIQK²⁹⁰ from probable integral membrane protein Cj0587. D, ETD-MS of the glycopeptide ²⁰⁵TIANDAYRENNHTK²¹⁸ from putative sulfatase Cj0256. See supplemental Table S2 and supplementary data for all annotated spectra. ETD is dominated by charge-reduced species and multiple ions corresponding to neutral loss species (denoted with &). Intens, intensity.

Fig. 4.

Novel C. jejuni glycosylation sites identified using HCD- and ETD-based approaches. HCD-MS (A) and ETD-MS (B) of the glycopeptide ⁷⁰NCGDFNK⁷⁶ from putative lipoprotein Cj0089c are shown. C, HCD-MS of the glycopeptide ²⁸²DNNLSLIQK²⁹⁰ from probable integral membrane protein Cj0587. D, ETD-MS of the glycopeptide ²⁰⁵TIANDAYRENNHTK²¹⁸ from putative sulfatase Cj0256. See supplemental Table S2 and supplementary data for all annotated spectra. ETD is dominated by charge-reduced species and multiple ions corresponding to neutral loss species (denoted with &). Intens, intensity.

Fig. 5.

Comparison of glycopeptide identifications from whole cell lysates and membrane protein-enriched fractions.

Fig. 6.

CID-MS of SBA-bound, 2-DE-separated, trypsin/proteinase K-digested, and graphite-purified glycopeptide (⁹⁷DANLT¹⁰¹) from secreted transglycosylase (Cj0843c). See supplemental Table S2 and supplemental data for all annotated spectra.

Fig. 7.

Predicted subcellular localization of glycoproteins from C. jejuni HB93-13 identified in this study.

Fig. 8.

Functional classification of identified C. jejuni glycoproteins. 34% of identified proteins have no known function with the largest group of predicted functions belonging to membrane transport (21%; 11 proteins).