Shotgun proteomics

All samples were reconstituted in 0.1% formic acid and separated using an Easy-nLC1200 liquid chromatograph (Thermo Scientific) followed by peptide detection on a Q Exactive HF mass spectrometer (Thermo Scientific). Samples were separated on a 75 µm x 15 cm custom-made fused silica capillary packed with C18AQ resin (Reprosil-PUR 120, 1.9 µm, Dr. Maisch), flow rates and gradients were adjusted according to the experiment. Except for plasma membranome analysis, peptide mixtures were separated on a 35 min acetonitrile gradient in 0.1% formic acid at a flow rate of 400 nl/min (5%–38% ACN gradient for 23 min, 38%–60% ACN gradient for 3 min, 60%–95% ACN gradient for 2 min). For plasma membranome analysis, peptide mixtures were separated on a 75 min acetonitrile gradient in 0.1% formic acid at a flow rate of 400 nl/min (7%–38% ACN gradient for 53 min, 38%–60% ACN gradient for 5 min, 60%–100% ACN gradient for 5 min). Peptides were identified in full MS / dd MS² (Top15) mode, dynamic exclusion was enabled for 20 s and identifications with an unassigned charge or charges of one or >8 were rejected.