Microqtof qiii mass spectrometer

For LC-MS, an aliquot of ANR (20 μl, 80 μm [4Fe-4S]) was combined with an equal volume of oxygenated buffer (∼220 μm O₂) or anaerobic buffer and allowed to react for 15 min. Samples were diluted to 2.9 μm final concentration, with an aqueous mixture of 1% (v/v) acetonitrile, 0.3% (v/v) formic acid, sealed, removed from the anaerobic cabinet, and loaded (5 μl) onto a ProSwift RP-1S column (4.6 × 50 mm) (Thermo Scientific) on a Ultimate 3000 UHLPC system (Dionex, Leeds, UK). Bound protein was eluted (0.2 ml/min) using a linear gradient (15 min) from 1% to 100% (v/v) acetonitrile, 0.1% (v/v) formic acid. The eluent was continuously infused into a Bruker microQTOF-QIII mass spectrometer, running Hystar (Bruker Daltonics, Coventry, UK), using positive mode electrospray ionization. Compass Data Analysis with Maximum Entropy v1.3 (Bruker Daltonics, Coventry) was used for processing of spectra under LC peak. The mass spectrometer was calibrated with ESI-L tuning mix (Agilent Technologies).

UV–visible absorbance measurements were made with a Jasco V550 spectrometer. The extinction coefficient for the E. coli [4Fe–4S] FNR (ε_{406 nm} = 16,200 M⁻¹ cm⁻¹ [28 (link)]) was used to calculate the amount of [4Fe–4S] cluster present in FnrP samples. CD spectra were measured with a Jasco J810 spectropolarimeter. For liquid chromatography–mass spectrometry (LC–MS) an aliquot of FnrP (100 μL, 46 μM [4Fe–4S]) was combined with varying aliquots of aerobic (229 μM O₂, 20 °C) or anaerobic assay buffer (200 μl final volume), and allowed to react for 15 min. Samples were diluted to ~2 μM final concentration, with an aqueous mixture of 1 % (v/v) acetonitrile, 0.3 % (v/v) formic acid, sealed, removed from the anaerobic cabinet and analyzed by an LC–MS instrument consisting of an Ultimate 3000 UHLPC system (Dionex, Leeds, UK), a ProSwift RP-1S column (4.6 × 50 mm) (Thermo Scientific), and a Bruker microQTOF-QIII mass spectrometer, running Hystar (Bruker Daltonics, Coventry, UK), as previously described [9 (link)].

SDS–PAGE gels and on-cell assay data was collected using BioRad Gel Doc XR+ gel imager, BioRad ChemiDoc XRS+ gel imager and GE ImageQuant LAS 4000 gel imager. On-cell assay quantitative data were collected using the CLARIOstar plate reader (BMG labtech). MS data were collected using a Bruker microQTOF-QIII mass spectrometer. LC–MS/MS measurements were performed on an Orbitrap Eclipse TribridTM mass spectrometer (Thermo Fisher Scientific) equipped with an UltiMate 3000 RSLCnano System (Thermo Fisher Scientific) using a nanoEase M/Z column (HSS C18 T3, 100 Å, 1.8 μm; Waters). For SDS–PAGE analysis, we used Image Lab (v.6.1.0, build 7). For DNA sequence analysis, we used Staden 2.0.0b11-2016 and SnapGene. For graph generation, we used Microsoft Excel (v.2210, build 16.0.15726.20188) and Graphpad Prism 9. MS/MS data analysis was performed using Scaffold v.5.1.2 (Proteome Software Inc.). Protein structure images were produced using UCSF chimera 1.16. Routine liquid chromatography–MS analysis was carried out as previously described^{51 (link)}.