Empore octadecylc18

100 µg protein lysates from each sample were reduced with 10 mM DTT for 45 min at 37 °C and alkylated with 55 mM chloro-acetamide for 30 min at room temperature in the dark. Samples were diluted with 5 volumes of 40 mM Tris–HCl, pH 7.6 and hydrolyzed with trypsin (Promega, Mannheim, Germany) in a 1:50 (w/w) enzyme-to-substrate ratio during overnight incubation at 37 °C in a thermoshaker at 700 rpm. Samples were acidified with formic acid (FA) to a concentration of 0.5% (v/v). Samples were desalted using self-packed stage-tips [10 discs, Ø 1.5 mm, C18 material, 3 M Empore™ Octadecyl C18, Saint Paul, MN, USA; wash solvent: 0.1% formamide (FA); elution solvent: 60% acetonitrile (ACN) in 0.1% FA]. 100 µg of protein hydrolysate were dissolved in 50 mM HEPES, pH 8.5 and mixed for 10 min at 20 °C. Tandem Mass Tag (TMT) reagents (Thermo Fisher Scientific) were added to each protein hydrolysate to a final concentration of 11.6 mM and incubated at 400 rpm on a thermomixer for 1 h at 20 °C. Reactions were stopped with 0.4% hydroxylamine (v/v). Labelled peptide solutions were pooled and desalted on Sep-Pak tC18 RP extraction cartridges (Waters Corp., Finglas, Ireland; wash solvent: 0.1% FA; elution solvent: 60% ACN in 0.1% FA). TMT-labelled samples were fractionated using a Dionex Ultimate 3000 HPLC System (Dionex Corporation, Idstein, Germany) and collected in 32 fractions.