Itraq labeling

The iTRAQ Labeling procedures were conducted according to the manufacturer's instructions (AB SCIEX, Shanghai, China). 100ug proteins of each group were precipitated with fivefold acetone at -20℃ for 1 h. Then, the mixture was centrifuged for 10min at 12,000 x g at 4℃. After removal of the suspension, the protein was dried using a vacuum centrifuge. Then, the protein was resuspended in 50μl dissolution buffer, reduced by 4μl reducing reagent for 1 h at 60℃, and then alkylated by 2μl cysteine blocking reagent for 10 min at room temperature. Protein samples were then digested with 50μl trypsin (50ng/μl) at 37℃ for 12 h. Tryptic peptides were dried by vacuum centrifugation and labeled with the iTRAQ regents at room temperature for 2 h. Afterward, 100μl distilled water was added to stop the reaction, the samples were mixed with equal amounts, and then the dried samples using a vacuum centrifuge were left for isolation and identification.

Peptide mixtures were desalted using in-house-packed stage tip columns composed of two C18 membrane disks (Empore 3 M, Bellefonte, USA) and porous R2/R3 reversed-phase resins (Thermo Fischer Scientific). In brief, samples were acidified to pH ~ 2 before peptides were applied to 0.1% TFA pre-equilibrated columns, washed with 0.1% TFA, and eluted using 70% ACN, 0.1% TFA. The eluted peptides were vacuum centrifuge dried before being reconstituted in 0.05 M TEAB prior to amino acid analysis (AAA) to measure peptide concentration. AAA was performed by lyophilizing a small aliquot of peptide sample and adding 200 µL hydrolysis buffer (6 M HCl, 0.1% phenol, 0.1% thioglycolic acid), filling with argon and subsequently evaporating under vacuum. Samples were then incubated at 110 °C ON. After hydrolysis, the amino acids were analyzed on a Biochrom30 amino acid composition analyzer (Cambridge, UK) as described in^{38 (link)}, and 20 µg of peptides from each sample were then transferred to new Eppendorf tubes, vacuum dried, and re-dissolved in 0.5 M TEAB prior to iTRAQ labeling (AB Sciex), as described by the manufacturer. After iTRAQ labeling and pooling, samples were vacuum centrifuge dried, re-dissolved in 0.1% TFA, and desalted on in-house-packed R2/R3 stage tip columns, as previously described.