Plastoglobules BN-PAGE experiment (Arabidopsis PGs under unstress and high light conditions)

Proteome comparison of isolated plastoglobules (PGs) from Arabidopsis thaliana (Col-0 ecotype) under unstress (UNS) and high light (HL) stress conditions. For both conditions, PGs were isolated from three independent set of plants each (i.e. three for UNS and three for HL). PGs were isolated from the plants when they were 4 weeks old under long day photoperiod (16 hours of light and 8 darkness). The light stress was applied to the designated set of plants 5 days prior the completion of the 4 weeks by shifting the light intensity from 120 uE to 500 E. The PGs were solubilized with 1.1 % n-Dodecyl--D-Maltoside and separated in a Blue-Native gel. After the electrophoretic separation, gel lanes were cut and further divided into 10 independent slices corresponding to different molecular weights. These gel slices were subjeted to in-gel digestion with trypsin and the resulting peptides labeled with TMT reagents. Labeled peptides were mixed 1:1 and purified by solid phase extraction using c18 StageTips. Eluted peptides were sprayed into a ThermoScientific Q-Exactive HF-X mass spectrometer. Survey scans were taken in the Orbi trap (120000 resolution, determined at m/z 200) and the top 20 ions in each survey scan are then subjected to automatic higher energy collision induced dissociation (HCD) with fragment spectra acquired at 45000 resolution. Additionally, PG samples that were not separated in a Blue-native gel were trypsin digested and subjected for mass-spectrometry analysis as previously described.