Masslynx v4

To evaluate the K_d of the IscSU complex by native mass spectrometry, an aliquot of purified protein (as isolated IscU or IscS) was first exchanged into 250 mM ammonium acetate pH 8.0 using desalting columns (PD MiniTrap G25, Cytiva). The protein concentration was determined by absorbance and working solutions (200 μL) were prepared by combining aliquots of IscS and IscU to achieve the desired concentration or IscS/IscU ratio, then infused directly into the source of a Waters Synapt XS (Waters Inc.) mass spectrometer operating in the positive ion mode. Data were acquired and processed with Mass Lynx v4.2 (Waters Inc.) over the m/z range of 3000 to 8000 m/z with parameters as follows: dry gas flow 4 L min⁻¹, nebuliser gas pressure 6 Bar, dry gas 150 °C, source temperature 80 °C, capillary voltage 3100 V, offset 30 V, cone voltage (isCID) 150 V, trap collisional energy 10 eV, and transfer collision energy 2 eV. Spectra were averaged, and the neutral mass spectrum calculated (90–125 kDa) using the MaxEnt1 deconvolution algorithm of MassLynx v4.2 (Waters Inc.). At least two datasets were acquired, expressed as fractional saturation, and then averaged prior to fitting with Dynafit (Biokin), as previously described.^{22 (link)} The mass spectrometer was calibrated with sodium iodide. Average resolution for the measured range was 20 000 FWHM.

The LC-MS system utilized for metabolomic analysis was composed of a Waters Acquity I-Class PLUS ultraperformance liquid chromatography system coupled to a Waters Xevo G2-XS QTof high-resolution mass spectrometer. The chromatographic column used was an Acquity UPLC HSS T3 column (1.8 µm, 2.1 mm × 100 mm) purchased from Waters. The mobile phase consisted of aqueous formic acid solution (A) and acetonitrile (B), and the gradient elution program was as follows: 0 min, 98%; 0.25 min, 98%; 10 min, 2%; 13 min, 2%; 13.1 min, 98%; 15 min, 98% and 0 min, 2%; 0.25 min, 2%; 10 min, 98%; 13 min, 98%; 13.1 min, 2%; and 15 min, 2%. The Xevo G2-XS QTof high-resolution mass spectrometer can collect primary and secondary mass spectral data in MSE mode under the control of acquisition software (MassLynx V4.2, Waters). In each data acquisition cycle, dual-channel data acquisition with low collision energy and high collision energy can be carried out simultaneously. The low collision energy was 2 V, the high collision energy was 10–40 V, and the scanning frequency was 0.2 s. The parameters of the electrospray ionization (ESI) source were as follows: capillary voltage, 2000 V (positive ion mode) or −1500 V (negative ion mode); taper hole voltage, 30 V; ion source temperature, 150°C; desolvation gas temperature, 500°C; back blowing flow rate, 50 L/h; and flow rate of desolvation gas, 800 L/h.