Quantitative Proteomics of Plant Flowers

Three independent biological replicates performed in our experiment. Three internal standards (IS-1, IS-2, and IS-3) were prepared by mixing one biological replicate from the six tested samples. Protein extraction performed using TCA (trichloroacetic acid)-acetone method with some modifications.^{41 (link)} Flowers were ground to powder in liquid nitrogen using a mortar and pestle. The powder was mixed with cold acetone which contains 10% (w/v) TCA and 0.07% (w/v) 2-mercaptoethanol and then precipitated for 2 h at −20 °C. After centrifuging at 25 000 × g for 30 min, the pellet was washed with cold acetone twice and suspended in buffer containing 8 M urea, 4% CHAPS, 2% ampholyte and 20 mM DTT. After centrifuging at 25 000g for 60 min, the supernatant underwent a reductive alkylation reaction. The concentration of the protein solution was determined using the Bradford method with BSA as a standard. For protein digestion, 100 μg of protein sample was digested with trypsin at 20 : 1 (w/w) for 4 h at 37 °C and then digested 8 h at 37 °C after adding the same amount trypsin. Sample digestions were vacuum dried and reconstituted with 0.5 M TEAB. iTRAQ labeling was performed using iTRAQ 8-plex kits (AB Sciex, USA) according to the manufacturer's protocol.^{42 (link)}