The isolates were cultured on Luria Bertani agar overnight. Subsequently, a single colony from each isolate was cultured in Mueller-Hinton broth. An optimization step to establish optimum bacterial growth condition to obtain the highest number of identifiable proteins was also performed. The number of identified proteins from an overnight bacterial culture and from bacteria harvested at 0.5 of OD600 were compared. In the overnight culture, outer membrane proteins were abundant, reducing the number of other identified proteins. A bacterial culture harvested at OD600 ~0.5 thus provided a better sample for protein analysis (data not shown). Therefore, the culture of E. coli in Mueller-Hinton broth without or with appropriate antibiotics for 4 hours with shaking at 250 rpm at 37oC to reach the exponential growth phase at OD600 of 0.5 was used.
Proteins were quantified in triplicates using the BCA assay (Sigma-Aldrich) and bovine serum albumin (BSA) as standard. After incubation at 37oC for 30 min, a microplate reader (BMG Labtech, Ortenberg/Germany) was used to determine the protein concentration at 562 nm.
Reduction and alkylation:
Aliquots of 1 μg protein/μL were mixed with ammonium bicarbonate (100 μl of 50 mM) for 10 s. Ten microliters of 20 mM DTT/bicarbonate were added to each sample and incubated for 1 h at 60oC. Iodoacetamide (10 μl of 1 M in 100 mM bicarbonate) was added to each tube and incubated for another hour at 37oC, protected from light.
Trypsin digestion and desalting:
Samples were digested with Trypsin Gold (Promega, USA) to cleave proteins into peptides on the carboxyl side of amino acid residues lysine and arginine. Following digestion, 0.1% formic acid (100 μL) was added and samples were centrifuged (15,000 rpm for 15 min) using a 10 kDa size exclusion membrane (PALL, Nanosep Cheltenham Vic, Australia). The flow-through was retained for a desalting step using a 3 mm piece of an Empore C18 (Octadecyl) SPE Extraction Disk. The disk was excised and placed in a gel loader tip and 5 μL of a POROS R3 slurry were added to form a micro-column. This column was washed with trifluoroacetic acid (20 μL, 0.1% in water). Peptides were eluted from the micro-column by three washes of acetonitrile (20 μL, 0.1% formic acid). Elutes were pooled and samples were dried at room temperature in a vacuum evaporator for 45 min. Subsequently, the samples were reconstituted with 100 µl of 0.1% formic acid in H2O and centrifuged for 2 min at 10,000g to remove particulates.
Sample analysis by LC-MS/MS
LC-MS/MS analysis of digested E. coli lysates was performed on a Tandem Quadrupole Time-of-Flight mass spectrometer Sciex TripleTOF 5600 (Sciex) coupled to an Eksigent 1D+ Nano LC system and a nanoFlex cHiPLC system (Eksigent) with a Nanospray III Ion Source (Sciex). Peptides were separated using a linear gradient (60 min for 5 to 80% B at 500 nL/min) of 0.1% formic acid in water and 0.1% formic acid in acetonitrile, and were delivered by a nanospray III electrospray interface (105 mm stainless steel emitter, Thermo Fisher THIES528).
Data acquisition of peptide separation using LC-MS/MS was performed using two different methods: the information-dependent acquisition (IDA) method and the data-independent (SWATH) acquisition method. Technical duplicates were performed for the IDA method.
Following the LC-MS/MS analysis, the mass spectral data generated from the IDA method were processed through two different search algorithms, Mascot (Matrix Science, v. 2.4.0) and Paragon (ABSciex, ProteinPilot Software v. 22.214.171.124, 1654). To generate an ion library, LC-MS/MS mass spectral data were firstly analysed using Mascot and the Eubacteria database from SwissProt for a qualitative analysis to identify and detect the presence or absence of the proteins of interest. Secondly, the analysis using Paragon involved a search against a suitable FASTA-formatted E. coli protein database from UniProt for the identification of peptides from the mass spectral data54. The data from Paragon were loaded onto PeakView (Sciex, v.126.96.36.199) to interrogate the SWATH data bank using the ion library generated in ProteinPilot. PeakView performs targeted and non-targeted data processing and generates extracted ion chromatograms (XIC). The data were then transferred to Markerview (Sciex, v. 188.8.131.52) for result interpretation and quantitative analysis. Markerview allows for a rapid review of the data to determine up and down-regulation of protein expression26. The data were processed using principal component analysis (PCA), which is an unbiased multivariate statistical analysis method that compares data across multiple samples, revealing groupings among data sets and graphically present the groupings in a Scores plot.
Specific to analyse the data from the SWATH method, three different software programs were used. Firstly, Paragon was used to streamline protein identification and quantitation by identifying hundreds of peptide modifications and non-tryptic cleavages simultaneously to build an ion library. Then, using PeakView (Sciex, v. 184.108.40.206), the SWATH data were interrogated against the ion library. PeakView allows mass spectral data to be explored and interpreted for processing accurate mass data, structural interpretation and batch analysis. MarkerView was then used to review the data to determine the up or down regulation of protein expression in the bacterial samples through the use of Principle Components Analysis (PCA), a statistical tool that produces a visual representation of patterns in a dataset. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE55 partner repository with the dataset identifier PXD008019.