Cyclotrons

M. smegmatis cells expressing different M. tuberculosis proteins were grown in identical conditions to late log phase or stationary phase. In all expression cultures the ZYP–5052 autoinduction media was used for F₄₂₀ production experiments and the media to flask volume ratio was kept constant at 20%. In order to optimize the media for F₄₂₀ production, the ZY component of ZYM–5052 media was replaced by commonly used media bases including 2× ZY, YT (0.8% tryptone, 0.5% yeast extract and 42.77 mM NaCl), TB (1.2% tryptone, 2.4% yeast extract and 0.4% glycerol), SOB (2% tryptone, 0.5% yeast extract, 8.56 mM NaCl, 2.5 mM KCl and 10 mM MgCl₂) and SOC (SOB with 20 mM glucose). Iron and sulphur supplements (ferric ammonium citrate, ferric citrate and ferrous sulphate all at 0.1 mg/mL and L–cysteine at 1 mM) were also added to the expression media as a possible requirement for the FbiC enzyme. L–glutamate and manganese chloride (1 mM final concentration) were also added to the expression media to evaluate their necessity for FbiB–mediated F₄₂₀ production [22] (link).
To ascertain the optimum growth period for F₄₂₀ production, eight identical cultures of M. smegmatis cells expressing the recombinant FbiABC construct were set up. Each culture had a wild type M. smegmatis culture as a control. At 24 h intervals, one culture each of control and recombinant FbiABC–expressing M. smegmatis cells were harvested and processed to monitor the F₄₂₀ production level. The procedure was carried out for eight days and the F₄₂₀ production ratio for each day was calculated by dividing the F₄₂₀ fluorescence from FbiABC–expressing cells by fluorescence of the wild type control.
M. smegmatis cells were centrifuged for 15 min at 16000×g and the resulting media were used for FO characterization. The cell pellets were washed with 25 mM sodium phosphate buffer, pH 7.0 and were subsequently resuspended in 1 mL of the same buffer per 100 mg of cells (wet weight). The cell suspensions were autoclaved at 121°C for 15 min to break the cells open and were then centrifuged for 15 min at 16000×g. Fluorescence of the media and the extract were monitored using excitation wavelength of 420 nm (405±10 nm filter) and emission wavelength of 480 nm (485±15 nm filter). All fluorescence experiments were performed using an EnVision Multilabel plate reader (Perkin Elmer) in a 96–well plate format and were carried out in triplicate.
The autoclaved cell extracts were further purified using a HiTrap QFF ion exchange column (GE Healthcare) to separate the intracellular FO from the F₄₂₀. The extract was run on the column pre–equilibrated with 25 mM sodium phosphate buffer, pH 7.0 and was subsequently washed with five column volumes of buffer. Two yellow fractions were eluted at 200 and 500 mM NaCl, respectively. The purified fractions were used for mass spectrometry analysis, together with the media from the previous step. The media (1 mL) was treated with an equal volume of cold acetone to precipitate the protein and the solution was then evaporated down to <0.5 mL to drive off the acetone. A mix of water and 5% aqueous methanol with 0.1% formic acid was added to bring the final concentration of methanol to less than 1% (total volume 4 mL). All samples were then applied to a pre–equilibrated Alltech Maxi–Clean 300 mg large pore 100Å C–18 SPE cartridge and washed with 4 mL 5% methanol containing 0.1% formic acid followed by 4 mL 10% methanol. Compounds were eluted with 4 mL 80% methanol containing 5 mM ammonium bicarbonate pH 8.5. Eluates were evaporated under nitrogen and redissolved in 80% methanol and 20 mM ammonium acetate ready for mass spectrometry. Samples were infused at 3 µL/min under negative electrospray conditions into an LTQ–FT mass spectrometer (Thermo Scientific). The ion intensity data were obtained using a source voltage of 2.5 kV and capillary temperature of 225°C. Ions were examined in both the ion trap and ion cyclotron resonance cells, the latter to obtain high resolution (100,000 at m/z 400) accurate mass data. This was necessary to confirm the atomic composition of the ions and help deconvolute the contribution of metal ion adducts (Na⁺/K⁺) to the levels of individual poly–glutamate species. Up to four sodium ions were adducted to produce some double charged negative ions.

Reagents used in the experiments were obtained from Sigma Aldrich, unless otherwise stated. Protected amino acids were purchased from Trimen Co., (Lodz, Poland). Concentrated solutions of peptides were made in ultrapure water (1 mM) and kept at—20°C until use.
Analytical and semi-preparative RP HPLC was performed using Waters Breeze instrument (Milford, MA, United States) with dual absorbance detector (Waters 2,487) on a Vydac C₁₈ column 5 μm, 4.6 × 250 mm, flow rate 1 mL/min, 50 min linear gradient, and a Vydac C₁₈ column 10 μm, 22 × 250 mm, flow rate 2 mL/min, 20 min linear gradient, respectively, from water/0.1% (v/v) TFA to 80% acetonitrile/20% water/0.1% (v/v) TFA. ESI-MS spectra were obtained on an FTICR (Fourier transform ion cyclotron resonance) Apex-Qe Ultra 7 T mass spectrometer (Bruker Daltonics, Bremen, Germany) equipped with standard ESI source. The instrument was operated in the positive-ion mode and calibrated with the Tunemix™ mixture (Agilent Technologies, Palo Alto, CA, United States). Solutions of peptides were introduced at a flow rate of 3 μL/min.

Affinity captured PSKH2-STREP samples were diluted 10-fold in 25 mM ammonium bicarbonate (pH 8.0), reduced with dithiothreitol and alkylated with iodoacetamide as described [73 (link)], 0.2 µg trypsin gold (Promega) was added and incubating at 37°C with gentle agitation for 18 h. Digests were then subjected to strong cation exchange using in-house packed stage tips, as previously described [74 (link)]. Dried peptides were solubilised in 20 µl of 3% (v/v) acetonitrile and 0.1% (v/v) TFA in water, sonicated for 10 min, and centrifuged at 13 000×g for 15 min at 4°C prior to reversed-phase HPLC separation using an Ultimate3000 nano system (Dionex) over a 60 min gradient [73 (link)]. All data acquisition was performed using a Thermo Orbitrap Fusion Lumos Tribrid mass spectrometer (Thermo Scientific), with higher-energy C-trap dissociation (HCD) fragmentation set at 32% normalised collision energy for 2+ to 5+ charge states. MS1 spectra were acquired in the Orbitrap (60K resolution at 200 m/z) over a range of 350 to 1400 m/z; AGC target = standard, maximum injection time = auto, with an intensity threshold for fragmentation of 2 × 10⁴. MS2 spectra were acquired in the Iontrap set to rapid mode (15 K resolution at 200 m/z), maximum injection time = 50 ms with a 1 min dynamic exclusion window applied with a 0.5 Da tolerance. For binding partner analysis, data was searched twice (search settings were identical, with the addition of low abundance resampling imputation of missing values in the second search) using Proteome Discoverer 2.4; searching the UniProt Human Reviewed database (updated weekly) with fixed modification = carbamidomethylation (C), variable modifications = oxidation (M), instrument type = electrospray ionisation–Fourier-transform ion cyclotron resonance (ESI-FTICR), MS1 mass tolerance = 10 ppm, MS2 mass tolerance = 0.5 Da. Percolator and precursor ion quantifier nodes (Hi3 Label free Quantification (LFQ)) were both enabled. All data was filtered to a 1% False discovery rate. Data from the first search was put into a custom R script that extracted all protein accessions with LFQ data in at least two of three replicates of a condition which was used to filter the imputation containing dataset. Accessions were used to obtain the gene name using the UniProt ID retrieval tool. Fold changes and T-tests were calculated and log₂ transformed, before importing into a custom R script for plotting. For PSKH2 tag orientation plotting, LFQ data was normalised to the level of PSKH2 for that replicate prior to calculations. Data was additionally analysed through PEAKS Studio (version XPro) using the same database, mass tolerances and modifications [74 (link)]. PEAKS specific search settings: instrument = Orbi-Trap, Fragmentation = HCD, acquisition = DDA, De Novo details = standard and a maximum of five variable PTMs possible. PEAKS PTM mode was enabled and filtering parameters of De Novo score >15, −log₁₀P(value) >30.0, Ascore >30.0 and seen in at least two of three replicates were applied for a PTM to be maintained. Filtered data are presented in Supplementary Table S1