Protein extraction, digestion, and tandem mass tag (TMT) labeling
TMT proteome of chloroplasts was adopted to identify differential proteins for achieving high-throughout and high-resolution screening. Chloroplasts were isolated from Z. marina leaves, which were dark-adapted (Dark) and exposed to 300 µE for 3 h (Light). The chloroplasts were suspended in BPP and Tris-saturated phenol, and then vortexed for 10 min. Proteins in the phenol phase collected via centrifugation at 12,000 g for 20 min were precipitated with ice-cold ammonium acetate, and then washed three times with 90% cooled acetone. The precipitate was dissolved in protein lysate (8 M urea, 1% SDS containing protease inhibitor), and the solution was then centrifuged as above to collect the protein supernatant. The above operations were performed at 4 °C. The protein concentration was measured by BCA.
Protein digestion was performed according to the standard procedure and the resulting peptide mixture was labeled using the 6-plex TMT reagents (ThermoFisher Scientific, Waltham, MA, USA). The peptides from Dark and Light samples were both labeled with TMT tags for three biological replicates. Briefly, each tube with 100 μg protein, containing 10 mM tris-(2-carboxyethyl)phosphine hydrochloride, was incubated at 37 °C for 60 min. The appropriate volume of iodoacetamide was added to a final concentration of 40 mM, and the solutions were incubated for 40 min in the dark. The samples were mixed with six volumes of cold acetone and the precipitates collected by centrifugation at 10,000 g for 20 min were resuspended with 100 µl 50 mM triethylammonium formate buffer. Trypsin solution was added to each sample tube according to a proportion of 1 : 50 (trypsin : protein) and the tubes were incubated overnight at 37 °C. The TMT reagent was added to corresponding peptide mixture respectively. All samples were then combined and vacuum dried.