Synapt g2 s quadrupole time of flight mass spectrometer

Pellet containing RNase digestion products is resuspended in 3 µL of milliQ water and separated on an Acquity peptide BEH C18 column (130 Å, 1.7 µm, 75 µm × 200 mm) using a nanoAcquity system (Waters). The column was equilibrated in buffer A containing 7.5 mM TEAA (Triethylammonium acetate), 7.0 mM TEA (Triethylammonium) and 200 mM HFIP (Hexafluoroisopropanol) at a flow rate of 300 nL/min. Oligonucleotides were eluted using a gradient from 15% to 35% of buffer B (100% methanol) for 2 min followed by elution with an increase of buffer B to 50% in 20 min. MS and MS/MS analyses were performed using SYNAPT G2-S (quadrupole time-of-flight mass spectrometer) from Waters Corporation. All experiments were performed in negative mode with a capillary voltage set at 2.6 kV and a sample cone voltage set at 30 V. Source was heated to 130°C. The samples were analyzed over an m/z range from 500 to 1500 for the full scan, followed by fast data direct acquisition scan (Fast DDA).

For GPX4^WT treated with the racemic mixture of ML162, different molar ratios of protein (in 50 mM Tris–HCl pH 8.0, 150 mM NaCl, 5 mM TCEP) and ligand were incubated at two different protein concentrations for up to 4 h (total volume of 15 µl per reaction). The protein concentration was adjusted to 10 and 100 µM, respectively, and the ligand was added in a fivefold, tenfold and 20-fold molar excess. The reaction was quenched by adding 1 µl 5%(v/v) trifluorocacetic acid (TFA) to a 15 µl reaction volume for LC-MS analysis.
The extent of covalent binding was assessed by LC-MS analysis using a Waters SYNAPT G2-S quadrupole time-of-flight mass spectrometer connected to a Waters nanoAcquity UPLC system. Samples were loaded onto a 2.1 × 5 mm MassPrep C4 guard column (Waters) and desalted with a short gradient (3 min) of increasing acetonitrile concentration at a flow rate of 100 µl min⁻¹. The spectra were analyzed using MassLynx version 4.1 and deconvoluted with the MaxEnt1 algorithm. Percent binding was determined using BiopharmaLynx (Waters).
For the GPX4^C66S mutant, a similar time-course experiment was carried out to follow the covalent reaction process. Here, the protein was tested at 50 µM with a 2.5-fold, fivefold and tenfold molar excess of the ligand and at 100 µM with a fivefold and tenfold molar excess.