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.