Ods c18 spe column

HL60 received RSL3 for 6 h to promote ferroptosis. THP-1 cells were pretreated with PMA (40 pmol/10⁶ cells) for 3 days and incubated with ferroptotic cells for 1.5 h at 37 °C. Unengulfed HL60 cells were removed. Then, the THP-1 cells were treated with 50 μM biotin or SAPE-biotin in RPMI 1640 (without FBS) for 4 h, and then harvested and incubated with prewashed streptavidin beads (Thermo Fisher Scientific) overnight at 4 °C on a shaker. Proteins interacted with SAPE-biotin were eluted by Laemmli buffer containing 500 μL 6 M urea, 25 μL 200 mM DTT, and 25 μL 500 mM IAA in dark at room temperature for 30 min. The eluent was incubated with 150 μL 2M urea, 150 μL 1 mM CaCl₂, and 1 μL trypsin (1 μg/μL) at 37 °C overnight. After that, the protein samples were purified by ODS C18 SPE column (Agilent) and analyzed by LC–MS/MS. Samples were then analyzed in a data-dependent acquisition mode by the LC−MS/MS, equipped with an EASY-nLC 1200 (Thermo Fisher Scientific) HPLC system and Orbitrap Fusion Lumos (Thermo Fisher Scientific) mass spectrometer. For LC separation, tryptic peptides were sequentially injected into an Acclaim PepMap 100 C18 column (100 μM × 2 cm, 5 μM, Thermo Fisher Scientific, P/N:164564) and an Acclaim PepMap 100 C18 column (50 μM × 15 cm, 2 μM, Thermo Fisher Scientific, P/N:164943).

PC12 cells were pretreated with the proteasome inhibitor MG132 (10 μM) for 4 hours after transfection with GPX4-FLAG plasmid for 24 hours. DAQ (DA + FeCl₃, 150 μM) was added 6 hours before harvest. The cell lysates were centrifuged at 12,000g for 10 minutes and the supernatant was mixed with the FLAG antibody overnight at 4°C on a shaker, before incubation with prewashed protein A/G agarose beads at 25°C for 4 hours. The beads were collected and washed, and the bound proteins were eluted by Laemmli buffer containing 500 μL 6 M urea, 25 μL 200 mM DTT, and 25 μL 500 mM IAA in the dark at 25°C for 30 minutes. The eluent was incubated with 150 μL 2 M urea, 150 μL 1 mM CaCl₂, and 1 μL trypsin (1 μg/μL) at 37°C overnight. After that, the protein samples were purified by ODS C18 SPE column (Agilent) and analyzed by LC-MS/MS, equipped with an EASY-nLC 1200 HPLC system and Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific). For LC separation, tryptic peptides were sequentially injected into an Acclaim PepMap 100 C18 column (100 μM × 2 cm, 5 μM, Thermo Fisher Scientific) and an Acclaim PepMap 100 C18 column (50 μM × 15 cm, 2 μM, Thermo Fisher Scientific).