Shotgun proteomics

Sample preparation for LC-MS/MS Eight million of Cy3/5-positive autophagosomes (roughly 10ug of proteins) were denatured with 2% sodium deoxycholate, 50 mM Tris-HCl pH 8.5, 2.5 mM TCEP, 10 mM chloroacetamide at 95°C for 10 min. Lysates were prepared with in-StageTip (iST) processing method for LC-MS/MS as previously described by Kulak et al. 2014. Briefly, Proteins were digested overnight at 37°C with 1 volume of 50mM Tris-HCl pH 8.5 containing LysC (Wako Chemicals) at 1:100 (w/w) ratio and Trypsin (Promega V5113) at 1:100 (w/w) ratio. Digestion was stoped with 2 volumes of 1% TFA in isopropanol. Digested peptides were purified with Empore SDB-RPS (styrenedivinylbenzene - reverse phase sulfonate) disks in stage tip (3M Empore) and were dried for LC-MS/MS. Dried peptides of each sample were resuspended in 2% (v/v) acetonitrile / 1% (v/v) formic acid solution. Peptides were separated with Easy nLC 1200 (ThermoFisher Scientific) using a 30 cm long, 75 μm inner diameter fused-silica column packed with 1.9 μm C18 particles (ReproSil-Pur, Dr. Maisch) and kept at 50 °C using an integrated column oven (Sonation). Individual peptides were eluted by a non-linear gradient from 4-28% acetonitrile over 120 min, followed by a step-wise increase to 76% acetonitrile in 6 min, which was kept for another 9 min and sprayed into a QExactive HF mass spectrometer (ThermoFisher Scientific). Full scan MS spectra (300-1,650 m/z) were acquired with a resolution of 60,000 at m/z 200, maximum injection time of 20 ms and AGC target value of 3 x 106. The 15 most intense precursors were selected for fragmentation (Top 15) and isolated with a quadrupole isolation window of 1.4 Th. MS2 scans were acquired in centroid mode with a resolution of 15,000 at m/z 200, a maximum injection time of 25ms, AGC target value of 1 x 105. Ions were then fragmented using higher energy collisional dissociation (HCD) with a normalized collision energy (NCE) of 27; and the dynamic exclusion was set to 25s to minimize the acquisition of fragment spectra of already acquired precursors.