Proswift rp 1s column

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)].

Approximately 0.5 cm³ of breast cancer and matching adjacent normal tissue biopsy material was homogenized in lysis buffer (1% Igepal, 300 mM sodium chloride, 100 mM Tris, pH 8.0) supplemented with protease inhibitor cocktail (Roche) using a bead beater (Precellys 24 bead‐beater, Bertin Technologies) five times for 10 s at 6500 rpm. Lysates were cleared by subsequent centrifugation steps at 300 × g for 10 min and then 20 000 × g for 60 min. One milligram per sample of human anti‐HLA class I antibody (W6/32, ATCC HB‐95) was bound and cross‐linked to 1 mL Protein A beads (GE Healthcare) and used for immunoprecipitation of HLA complexes as described previously.16 In brief, lysates were incubated with the antibody beads overnight at 4 °C and washed subsequently with 50 mM Tris, pH 8.0 containing first 150 mM, then 450 mM and finally 0 mM NaCl. Peptides were eluted with 5 mL of 10% acetic acid. Dried peptides were resuspended and injected onto a 4.6 × 50 mm ProSwift RP‐1S column (Thermo Fisher Scientific). Peptides were separated from larger complex components by elution using a 500 μL min⁻¹ flow rate over 10 min from 2 to 25% acetonitrile in 0.1% trifluoroacetic acid. Alternate fractions were pooled and two final fractions were analyzed by nano‐ultra performance liquid chromatography tandem mass spectrometry (nUPLC‐MS²).