Quantitative proteomics of the Neisseria gonorrhoeae cell envelope and membrane vesicles
Neisseria gonorrhoeae (GC) is a human-specific pathogen, and the agent of a sexually transmitted disease, gonorrhea. There is a critical need for new approaches to study and treat GC infections because of the growing threat of multidrug-resistant isolates and the lack of a vaccine. Despite the implied role of the GC cell envelope and membrane vesicles in colonization and infection of human tissues and cell lines, comprehensive studies have not been undertaken to elucidate their constituents. Accordingly, in pursuit of novel molecular therapeutic targets, we have applied isobaric tagging for absolute quantification coupled with liquid chromatography and mass spectrometry for proteome quantitative analyses. Mining the proteome of cell envelopes and native membrane vesicles revealed 533 and 168 common proteins, respectively, in analyzed GC strains FA1090, F62, MS11, and 1291.
Sample Processing Protocol
Proteins obtained from the cell envelopes and MVs extractions (100 μg and 40 μg, respectively) were precipitated overnight at -20°C in 90% acetone and washed twice with ice-cold acetone. iTRAQ (AB Sciex) labeling was performed according to the manufacturer’s recommendations. The following iTRAQ tags were used to label peptides in appropriate GC strains: 114 for FA1090, 115 for F62, 116 for MS11 and 117 for 1291. The reactions were carried out for 1 h at room temperature and were stopped by the addition of 250 μl of 0.1% Trifluoroacetic acid (TFA). Labeled peptides were pooled and purified on C18 column (Honeywell Burdick & Jackson). iTRAQ labeled peptides were separated by strong cation exchange (SCX) using a Paradigm MG4 HPLC (Michrom Bioresources/Bruker). Peptide separations were performed using a Zorbax300 SCX column (2.1 mm × 150 mm, Agilent Technologies) with an in-line guard cartridge. Peptides were eluted using SCX A and SCX B buffers with a flow rate of 200 μL/min using the following gradient: 2% B from 0 to 5 min, 8% B to 5.1 min, 18% B to 20 min, 34% B to 32 min, 60% B to 40 min, 98% B to 40.1 min and held to 50 min, 2% B to 50.1 min and held to the end of the run at 60 min. SCX A buffer contained 5 mM KH2PO4 pH 2.7, 30% (v/v acetonitrile, ACN) and SCX B buffer contained SCX A buffer with 500 mM KCl. Fractions were collected at 2 min intervals throughout the run and pooled to a reduced number of fractions for reversed-phase (RP-HPLC-MS) analysis based on ultraviolet absorption at 220 nm. The pooled fractions were dried in a vacuum concentrator. Each pooled fraction was reconstituted with 0.1% TFA (100 μL) and desalted using SepPak C18 cartridges (Waters). Peptides were eluted with 50% ACN/0.1% TFA and concentrated in a vacuum concentrator. Subsequently, peptides were separated using Eksigent nano-two-dimensional high-pressure liquid chromatography (2D HPLC, AB Sciex). In-line de-salting was accomplished using a reversed-phase trap column (100 μm × 20 mm) packed with Magic C18AQ (5-μm 200 Å resin; Michrom Bioresources) followed by peptide separations on a reversed-phase column (75 μm × 250 mm) packed with Magic C18AQ (5-μm 100 Å resin; Michrom Bioresources) directly mounted to the Dionex Probot MALDI Spotter (ThermoFisher Scientific). A 90 min gradient from 7% to 35% ACN in 0.1% TFA at a flow rate of 500 nL/min was used for chromatographic separations. The gradient of solvent B was as follows: 0 min, 5% B; 2 min, 7% B; 92 min, 35% B; 93 min, 50% B; 102 min 50% B; 103 min, 95% B; 108 min, 95% B; 109 min, 5% B; 129 min, 5% B; 130 min, 5% B. The column effluent was mixed in a micro Tee with matrix (5 mg/mL alpha-cyano 4-hydroxy cinnamic acid (Sigma-Aldrich) with 2% Ammonium Citrate (Sigma-Aldrich) and 40 fmol/uL Glu-Fibrinopeptide B (in-house synthesis) delivered at 1 uL/min. Fractions were spotted at 10 second intervals onto a stainless steel MALDI target plate (AB Sciex). MS and MS/MS spectra were acquired on an Applied Biosystems 4800 Proteomics Analyzer (TOF/TOF) (AB Sciex) in positive ion reflection mode with a 200 Hz Nd:YAG laser operating at 355 nm, and accelerating voltage with 400 ns delay. MS spectra were obtained with minimal laser energy in order to maintain the best resolution. MS spectra for the entire sample set were collected first, and on each sample spot MS/MS spectra were collected for the 20 most intense peaks above the signal-to-noise ratio threshold of 20 using a collision energy of 2 keV and air as the collision gas. Both MS and MS/MS data were acquired on the sample spots using an internal calibration with Glu-Fibrinopeptide B.
Data Processing Protocol
Data analysis was performed using the Paradigm search algorithm, which is a part of the ProteinPilot software (version 4.0) (AB Sciex) against the SwissProt target-decoy database for N. gonorrhoeae FA1090 with 1963 protein entries (downloaded on January 17, 2012). The user-defined search parameters were selected as follows: Sample Type: iTRAQ 4plex, Cys. Alkylation: MMTS, Digestion: Trypsin, Instrument: 4800, ID Focus: Biological Modifications, Search Effort: Thorough, FDR Analysis: Yes, User Modified Parameter Files: No. All other search parameters, such as number of missed and/or non-specific cleavages allowed and the mass tolerance of the precursor and fragment ions, are hard-coded into the ProteinPilot software for analyzing MALDI 4800 data. The ProGroup Algorithm built within ProteinPilot software was used to perform the statistical analysis on the identified peptides to determine the minimal set of identifications. Data were normalized by bias correction built in ProteinPilot.
Zielke RA, Wierzbicki IH, Weber JV, Gafken PR, Sikora AE. Quantitative proteomics of the Neisseria gonorrhoeae cell envelope and membrane vesicles for the discovery of potential therapeutic targets. Mol Cell Proteomics. 2014 May;13(5):1299-317 PubMed: 24607996