LC-MS/MS analysis for methionine oxidised proteome

HEK293T cells were grown in 6-well plates in DMEM supplemented with 10% FBS, 50 units/mL penicillin, and 50 μg/mL streptomycin under 37 °C and 5% CO₂ conditions. Grown cells were incubated with 5 μM Ir-OA for 1 h, and the samples were irradiated with LED array (400 nm, 3.75 mW cm⁻² for 1 min, 225 mJ cm⁻²). Note that four different samples with negative controls were prepared (#1: Ir-/hv-, #2: Ir-/hv + , #3: Ir + /hv-, #4: Ir + /hv+). Irradiated cells were lysed by RIPA buffer for 20 min at 4 °C. After centrifugation (16,000 × g, 10 min, 4 °C), the supernatant protein lysis solution was denatured and separated by SDS-PAGE gel electrophoresis. Protein loading quantity was quantified using the BCA assay, and 50 μg of protein was loaded for each of the samples. For whole protein analysis with ‘Shot-Gun’ method, gel electrophoresis was proceeded up to approximately 1 cm length of the lane. The gel was stained with Coomassie blue for 2 h and washed for 6–12 h with destaining solution (H₂O, methanol, and acetic acid in a ratio of 50/40/10% (v/v/v)). Each clearly stained lane was divided into six parts according to protein size; these were further chopped up to small cubic shapes (approximately 1 mm × 1 mm × 1 mm) for efficient in-gel digestion. Each of the six parts was transferred to 1.5 mL low binding microtubes (Eppendorf, Hamburg, Germany). Gels were washed with 150 μL triple distilled water for 5 min on the shaker (in triplicate). Next, 150 μL of 0.1 M ammonium bicarbonate (ABC) was added to the tubes and washed on the shaker for 5 min (in triplicate). The tubes were incubated with 1:1 mixture of 0.1 M ABC and acetonitrile on the shaker for 5 min (in triplicate). The above step was repeated with 100% acetonitrile. (in triplicate). The whole washing process with ABC solution to acetonitrile gradient was repeated once again. Residual solvent after removal of the final acetonitrile was completely dried with a speed-vac. After the drying process, a 150 μL solution mixture of 10 mM dithiothreitol (DTT) and 100 mM ABC for reduction was added into each microtube, and the mixture was incubated using the ThermoMixer (79 × g, 60 min, 56 °C) (Eppendorf, Hamburg, Germany). The above solution for reduction was replaced with a 150 μL solution mixture of 55 mM iodoacetamide (IDA) and 0.1 M ABC for protein alkylation and the tubes were shaken on ThermoMmixer (79 × g, 30 min, 25 °C) in the dark. After alkylation was completed, 100 mM ABC was added, and the tube was shaken for 5 min. Next, 0.1 M ABC was replaced with a 1:1 mixture of 0.1 M ABC and acetonitrile. The tubes were also shaken for 5 min and the mixture solution was substituted with only acetonitrile. ABC washing steps after alkylation were repeated once again. The last residual acetonitrile was removed using a micro-pipette and the tubes were strictly dried again by speed-vac. Then, 25 ng/μL Trypsin Gold mass spectrometry grade (V5280; Promega, WI, USA) in 0.05 M ABC was added to microtubes and incubated on the ThermoMixer (20 xg, 12–18 h, 37 °C). The microtubes were vortexed and supernatant was transferred to other new microtubes. The remaining gels in old tubes were washed with a 2:1 mixture of 5% formic acid and acetonitrile and the supernatant was transferred again to the same tubes. The supernatants were fully dried by using a speed-vac. Prepared peptide samples were analysed by Q Exactive Plus orbitrap mass spectrometry (Thermo Fisher Scientific, MA, USA) equipped with a nanoelectrospray ion source. To separate the peptide mixture, we used a C18 reverse-phase HPLC column (500 mm × 75 μm ID) with an acetonitrile/0.1% formic acid gradient from 2.4–28% for 150 min at a flow rate of 300 nL/min. For MS/MS analysis, the precursor ion scan MS spectra (m/z 400–2000) were acquired in the Orbitrap at a resolution of 70,000 at m/z 400 with an internal lock mass. The 20 most intensive ions were isolated and fragmented by high-energy collision induced dissociation (HCD). The experiment was performed in triplicate. LC-MS/MS data acquisition software Xcalibur (ver. 4.1.31.9) from Thermofisher Scientific was utilised.