Protein reduction, alkylation, isobaric labelling and sample cleanup

For digestion, 100 μg protein from each sample was first reduced with 10 mM DTT at 37 °C for 60 min and then alkylated with 25 mM iodoacetamide (IAM) at room temperature for 45 min in darkness. The protein pool of each sample was digested with Sequencing Grade Modified Trypsin with a ratio of protein:trypsin of 50:1 at 37 °C overnight and 100:1 for a second digestion for 4 h. After trypsin digestion, peptides were desalted using a Strata X SPE column, vacuum-dried, reconstituted in 25 μL 500 mM TEAB and processed according to the manufacturer’s protocol for the 8-plex iTRAQ kit (Applied Biosystems, Inc.)⁷¹. Briefly, one unit of iTRAQ reagent was added to the peptide solution after thawing and dissolving in 50 μL isopropanol. The peptide mixtures were incubated for 2 h at room temperature and then pooled and dried by vacuum centrifugation. The dried and labelled peptides were reconstituted with HPLC solution A (2% ACN, pH 10) and then fractionated by high-pH reverse-phase HPLC using a Waters Bridge Peptide BEH C18 (130 Å, 3.5 μm, 4.6 × 250 mm). Briefly, peptides were first separated by a gradient of 2% to 98% acetonitrile in pH 10 at a speed of 0.5 mL/min over 88 min into 60 fractions. The peptides were then combined into 20 fractions and dried by vacuum centrifugation. The peptide fractions were desalted using a Ziptip C18 according to the manufacturer’s instructions. The samples were dried under vacuum and stored at −20 °C until MS analyses were performed.