Symmetry c18 180 μm 20 mm trap column

HeLa cells were transfected with empty vector, MARCH1-Myc, or MARCH1ΔRING-Myc along with INSR-GFP and HA-Ubiquitin. Twenty-hour after transfection, cells were serum starved overnight. MG132 was added 4 h before lysis in Co-IP Buffer (Pierce). INSR-GFP was immunoprecipitated using anti-GFP antibody and eluted in 200 μl 0.2 M glycine, pH 2.5. The eluate was partially dried to ∼100 μl and precipitated with MeOH:CHCl₃:Water (4:1:3 ratio). The pellet was dried and reconstituted in 8 M urea, 0.4 M ammonium bicarbonate, reduced with DTT, and alkylated with iodoacetamide in the dark. The sample was then trypsin-digested overnight at 37 °C. The digested sample was injected onto a Q-Exactive Plus (Thermo Fisher) liquid chromatography–tandem mass spectrometry system equipped with a Waters Symmetry C18 180 μm × 20 mm trap column and a 1.7 μm, 75 μm × 250 mm nanoACQUITY UPLC column (37 °C) for peptide separation. Trapping was performed at 5 μl min⁻¹, 99% Buffer A (100% water, 0.1% formic acid) for 3 min. Peptide separation was performed with a linear gradient over 140 min at a flow rate of 300 nl min⁻¹. Collected data were processed using MASCOT Distiller and Search Engine (v.2.4), and modification sites were manually verified.