Proteomics of Campylobacter glycosylation
N-linked glycosylation is an essential virulence determinant in Campylobacter jejuni, the major causative agent of gastroenteritis in the developed world. Glycosylation is encoded by the pgl gene cluster which encodes for the biosynthesis and attachment of a conserved heptasaccharide glycan to proteins in the C. jejuni periplasm. Over 80 membrane-associated proteins have been identified, however the functional role played by glycan attachment is almost completely unknown. We used quantitative proteomics by label-based and targeted strategies to examine glycosylation negative C. jejuni in comparison to wild-type. These technical approaches were considered as ‘discovery’ (label-based) and ‘validation’ data sets in our subsequent analysis. Inclusion of a glycosylation restored strain enabled us to further exploit the proteomics data to exclude non-specific protein abundance changes that could be considered as off-target effects. These data have provided a reference set of changes associated with protein N-glycosylation that could subsequently be tested by phenotypic analysis to determine the role of this modification in Campylobacter biology.
Sample Processing Protocol
All MS experiments were performed on separate biological triplicates. Proteins were extracted and digested with trypsin. In label-based experiments, peptides were labelled with TMT (Thermo Scientific) according to the manufacturer’s instructions. Samples labelled by TMT were then combined and desalted using HLB cartridges. All peptide samples were separated offline by HILIC for fractionation. HILIC fractions were then separated and analysed by RPLC-MS/MS using an Easy 1200 LC coupled to a Q-Exactive HF mass spectrometer. For the validation cohort, DIA-SWATH MS; both libraries and SWATH were performed on an Ekspert 425 nanoLC coupled to a TripleTOF® 6600 mass spectrometer (SCIEX).
Data Processing Protocol
Data files from TMT experiments were processed in ProteinDiscoverer (v. 2.2) and searched against UniProt C. jejuni NCTC11168 database UniProt (UP000000799; organism ID 192222; release May 24, 2018 last modification; 1623 proteins) with the SequestHT algorithm. Search parameters were static modifications; carbamidomethyl (C), variable modifications; oxidation (M), TMT-6plex (peptide N-term, K) and using precursor and fragment ion tolerances of 20 ppm. Peptide level false discovery rate (FDR) were determined using Percolator (v. 2.08.01). Rank 1 peptide spectral matches (PSMs) corresponding to a 1% FDR were then exported, and reporter intensities normalized internally to total reporter ion signals across all channels. Peptides with ambiguous protein assignments were removed, as were peptides containing known N-glycosylation sites. Reporter signals for remaining PSMs were summed to find total reporter signals for identified proteins. For proteins with a minimum of 2 unique identified peptides, values were imported into Perseus (v. 22.214.171.124) for statistical analysis. SWATH library data files were processed in Protein Pilot (v. 5.0), searched against the UniProt C. jejuni NCTC11168 proteome (as above) using the Paragon algorithm. The group file was then imported into Peakview (v. 126.96.36.19991) using a total protein number corresponding to a global protein level FDR of 1%. SWATH files were processed against the resulting library using a peptide confidence threshold of 95%, 1% FDR and an extracted ion chromatogram (XIC) width of 20 ppm.
Cain JA, Dale AL, Niewold P, Klare WP, Man L, White MY, Scott NE, Cordwell SJ. Proteomics reveals multiple phenotypes associated with N-linked glycosylation in Campylobacter jejuni. Mol Cell Proteomics. 2019 PubMed: 30617158