Methods
Bacterial strains and culturing conditions : N. gonorrhoeae MS11 [55] and N. gonorrhoeae strains expressing chimeric pglO alleles [52] used within this study are listed in Supplementary Materials Table 1. N. gonorrhoeae strains were grown for 16-18hr (at 37oC with 5% CO2) on GC medium (Difco) supplemented with Kellogg’s supplement [56]. Cells were pelleted (4°C, 2800 g , 10 minutes), washed with ice-cold PBS (4°C, 10,000g ) and then snapped frozen.
SDS-PAGE and immunoblotting: SDS-PAGE and immunoblotting ofN. gonorrhoeae glycoproteins was undertaken as previously described [48]. Briefly, whole-cell lysates were prepared from equivalent numbers of cells by heating cell suspensions to 65oC in SDS–sample loading buffer and then lysates separated on a 15% SDS-PAGE gel prior to being transferred to a nitrocellulose membrane. Glycoproteins were detected by immunoblotting with the npg3 monoclonal rabbit antibody reactive to theN. gonorrhoeae di-N-acetylbacillosamine-galactose-galactose glycan [43] at a 1:10,000 dilution and detected using a alkaline phosphatase-coupled goat anti-rabbit secondary antibody (Sigma).
Proteome sample preparation: Snap frozen cells were resuspended in 4% sodium dodecyl sulfate (SDS), 100 mM Tris pH 8.5, and boiled at 95°C for 10 minutes with shaking (2000 rpm; Eppendorf ThermoMixer®) and ~1mg of protein precipitated by mixing with 4x volume of ice-cold acetone and incubating overnight at -20°C. Protein samples were then pelleted at 0°C, 4000 g , 10 minutes, the acetone discarded and samples air dried. Precipitated proteome samples were prepared using S-trap mini columns (Protifi, USA) according to the manufacturer’s instructions. Briefly samples were resuspended in 4% SDS, boiled and then protein amounts quantified using a BCA assay (Thermo Fisher Scientific). 200 μg of each sample was then reduced with 10mM DTT at 95°C for 10 minutes, allowed to cool to room temperature then alkylated with 40mM of Iodoacetamide for 30 minutes in the dark. Samples were then acidified with phosphoric acid to a final concentration of 1.2%, then mixed with seven volumes of 90% methanol/100mM TEAB pH 7.1 before being applied to S-trap mini columns. Samples were washed four times with 90% methanol/100mM TEAB pH 7.1 to remove SDS then Trypsin/Lys-c (1:100, Promega, USA) in 100mM TEAB pH 8.5 spun through the S-trap columns. Samples were digested overnight at 37°C then collected from the S-traps by washing with 100mM TEAB pH 8.5 followed by 0.2% FA followed by 0.2% FA/50% ACN. Peptide washes were pooled, dried and then resuspended in Buffer A* (0.1% TFA, 2% ACN) before being cleaned up with home-made high-capacity StageTips composed of 1 mg Empore™ C18 material (3M) and 5 mg of OLIGO R3 reverse phase resin (Thermo Fisher Scientific, USA) as previously described [57, 58]. Columns were wet with Buffer B (0.1% FA, 80% ACN) and conditioned with Buffer A* prior to use. Resuspended samples were loaded onto conditioned columns, washed with 10 bed volumes of Buffer A* and bound peptides were eluted with Buffer B before being dried then stored at -20˚C. For glycoproteomic analysis ofN. gonorrhoeae strains possessing chimeric pglO alleles, pooled references were created by mixing individual replicates of eachN. gonorrhoeae strain expressing a single chimeric pglOallele in equal amounts resulting in 15 pooled references in total, in line with the gas phase fractionation library approach of Pino et al. used for DIA analysis [53].
LC-MS FAIMS fractionation based glycoproteomics : Purified peptide samples of either biological replicates of N. gonorrhoeaeMS11 or the pooled references of N. gonorrhoeae strains expressing chimeric pglO alleles were re-suspended in Buffer A* and separated using a two-column chromatography set up composed of a PepMap100 C18 20 mm x 75 μm trap and a PepMap C18 500 mm x 75 μm analytical column (Thermo Fisher Scientific) coupled to a Orbitrap Fusion™ Lumos™ Tribrid™ Mass Spectrometer equipped with a FAIMS Pro interface (Thermo Fisher Scientific). 125-minute gradients were run for each sample altering the buffer composition from 2% Buffer B to 28% B over 106 minutes, then from 28% B to 40% B over 9 minutes, then from 40% B to 80% B over 3 minutes, the composition was held at 80% B for 2 minutes, and then dropped to 2% B over 2 minutes and held at 2% B for another 3 minutes. The Lumos™ Mass Spectrometer was operated in a FAIMS data-dependent mode with individual analytical runs collected at FAIMS Compensation Voltages (CVs) -20, -30, -40, -50, -60, -70 and -80 for N. gonorrhoeae MS11 as previously described [35] or FAIMS CVs -25, -35, -45, -55, -65 and -75 for N. gonorrhoeae strains possessing chimericpglO alleles. For each FAIMS CV, a single Orbitrap MS scan (350-2000 m/z , maximal injection time of 50 ms, an Automated Gain Control (AGC) of maximum of 4*105 ions and a resolution of 120k) was acquired every 3 seconds followed by Orbitrap MS/MS HCD scans of precursors (NCE 30%, maximal injection time of 80 ms, an AGC set to a maximum of 2*105 ions and a resolution of 30k). HCD scans containing the oxonium ions (204.0867; 138.0545, 366.1396 as well as the diNAcBac specific ions 229.1189 and 211.1082 m/z ) triggered three additional product-dependent MS/MS scans of potential glycopeptides; a Orbitrap EThcD scan (NCE 15%, maximal injection time of 300 ms, AGC set to a maximum of 2*105 ions with a resolution of 30k and using the extended mass range setting to improve the detection of high mass glycopeptide fragment ions [59]); a ion trap CID scan (NCE 35%, maximal injection time of 40 ms, an AGC set to a maximum of 5*104 ions) and a stepped collision energy HCD scan (using NCE 35% with 5% Stepping, maximal injection time of 300 ms, an AGC set to a maximum of 2*105 ions and a resolution of 30k).
LC-MS Data-independent acquisition (DIA) proteome analysis:Proteome samples from individual biological replicates of N. gonorrhoeae strains expressing chimeric pglO alleles were re-suspended in Buffer A* and separated using a Vanquish Neo (Thermo Fisher Scientific) equipped with a ACQUITY UPLC Peptide BEH C18 Column (300Å, 1.7 µm, 1 mm X 100 mm, Waters Corporation) coupled directly to an Orbitrap Exploris 480™ (Thermo Fisher Scientific). 5 μg of proteome digest was loaded directly on to the ACQUITY column for 1 minute at 50 μL/min with Buffer A (0.1% formic acid, 2% DMSO) and the buffer composition altered from 2% Buffer B (0.1% formic acid, 77.9% acetonitrile, 2% DMSO) to 26% B over 70 minutes, then from 26% B to 99% B over 2 minutes and then was held at 99% B for 1.5 minutes. The Orbitrap 480™ Mass Spectrometer was operated in a data-independent mode automatically switching between the acquisition of a single Orbitrap MS scan (350-951 m/z, maximal injection time of 50 ms, an AGC set to a maximum of 250% and a resolution of 120k) and the collection of 16 m/z DIA windows between 350 and 951 m/z (200-2000 m/z, NCE 28%, maximal injection time of 54 ms, a AGC set to 2000% and a resolution of 30k).
Glycoproteomic Data Analysis:FAIMS experiments were searched with FragPipe version 19 [60-64] using a modified “glyco-O-HCD” workflow allowing Carbamidomethyl as a fixed modification of cysteine in addition to oxidation of Methionine, N-terminal acetylation and the addition of the trisaccharide diNAcBac-Gal-Gal (mass 552.2167 Da) on Serines as variable modifications. Searches were performed against the N. gonorrhoeaeMS11 proteome (Uniprot Accession: UP000016457, 2047 proteins) allowing a 1% FDR. The resulting “psm.tsv” files for each FAIMS fraction were combined using R retaining only glycopeptides with a MSfragger Hyperscore >20. To further ensure high data quality assigned glycopeptides were manually assessed and the HCD and EThcD spectra assigned to the best scoring unique glycopeptide annotated with the Interactive Peptide Spectral Annotator [65]. Only glycopeptides which generated high confident HCD and EThcD MS/MS events, defined as spectra containing multiple b/y ions for HCD or c/z/y for EThcD as well as evidence of glycan oxoniums and characteristic loss of glycans within HCD spectra or the expected charge reduced masses for EThcD, were considered for further analysis. Manually validated glycopeptides are provided within Supplementary Data 1 and 2 and only these glycopeptides were used for analysis and data visualization.
DIA Proteomic analyses : DIA data were searched using Spectronaut (Biognosys, Switzerland) versions 17.1.221229 using the DIA-library free analysis workflow allowing oxidation of Methionine and N-terminal acetylation as variable modifications as well as Carbamidomethyl as a fixed modification of cysteine. The protein LFQ method set to MaxLFQ, single hit proteins excluded, and the precursor PEP cut-off was altered to 0.05 from the default 0.2 to improve quantitative accuracy. Searches were performed against the N. gonorrhoeae MS11 proteome (Uniprot Accession: UP000016457) supplemented with the chimeric PglO sequences. Protein outputs from Spectronaut were processed using Perseus (version 1.6.0.7) [66] with missing values imputed based on the total observed protein intensities with a range of 0.3 σ and a downshift of 1.8 σ. Statistical analysis was undertaken within Perseus by grouping biological replicates together which were assessed using ANOVA analysis to compare changes across all groups or using student t-tests to compare the proteome changes between the pglOmeningitidis group to all other combinations of pglO groups with a minimum fold change of +/- 1 considered for further analysis. Multiple hypothesis correction was undertaken using a permutation-based FDR approach allowing an FDR of 5%. Matching of protein homologs between N. gonorrhoeae MS11 and the reference N. gonorrhoeae FA 1090 strain (Uniprot accession: UP000000535) was undertaken using the Proteome comparison function of PATRIC, the bacterial bioinformatics database and analysis resource [67] with the N. gonorrhoeaeFA 1090 gene names, ORF assignments and Gene ontology information used to enable Enrichment analysis using Fisher exact tests in Perseus.
Data visualization and availability: Visualization of proteomic data was undertaken using R (version 4.2.1) using the tidyverse [68] collection of packages. All mass spectrometry data (RAW files, FragPipe outputs, Spectronaut experiment files, Rmarkdown scripts, and input tables) have been deposited into the PRIDE ProteomeXchange repository [69, 70]. All PRIDE accession numbers, descriptions of the associated experiments are provided within Supplementary Materials Table 2.