2.7. iTRAQ Labeling and Peptides Prefractionation by High pH Reverse Phase Chromatography

Peptides (100 μg) in 100 mM TEAB from each group were labeled using an 8plex iTRAQ reagents multiplex kit (ABI, Foster City, CA, USA), of which isobaric tags 113 and 114 were for the control group; 115, 116, and 117 were for 1 day postnoise exposure; and 118, 119, and 121 were for 7 days postnoise exposure. In brief, the 8plex iTRAQ reagents were first centrifuged at room temperature and reconstituted with 50 μl isopropyl alcohol to dissolve the iTRAQ labeling reagent. iTRAQ labeling reagents were added to the corresponding peptide samples and were allowed to react at room temperature for 1 h. A total of 100 μl of water was added to prevent the labeling reaction. One aliquot of each sample was analyzed by MS for the test of labeling efficiency. A total of eight sample groups were pooled and vacuum-dried. Each pool of mixed peptides was lyophilized and dissolved in solution A (2% acetonitrile, pH 10, pH adjusted with ammonium hydroxide). Samples were then loaded onto a reverse-phase column (C18 5 μm 4.6 × 250 mm, waters) and eluted using a step linear elution program: 5%–35% buffer B at flow rate of 0.7 ml/min (98% acetonitrile, without pH adjustment, solution B) for 30 min, 35%–95% buffer B for 2 min, 95% buffer B for 5 min, and 95%–5% buffer B for 2 min. Samples were collected every 1.5 min. The collected fractions (about 40) were finally combined into 10 pools and desalted on C18 Cartridges (Empore™ standard density SPE C18 Cartridges, bed I.D. 7 mm, 3 ml volume; Sigma, St. Louis, MO, USA).