2.3. Protein precipitation and trypsinization

Protein pellet were precipitated by acetone precipitation method. The precipitated pellet were dissolved in 10 μL of 8 M urea and the total volume was made to 15 μL with water. 20 mM DTT was added to samples for reducing them and heated for 15 min at 60 °C. Sample were cooled and 50 mM mM IAA (Iodoacetamide) was added to the samples to alkylate them and incubated for 15mins in the dark at RT. 82 μL of Ammonium Bicarbonate was added and proteins were digested by adding trypsin protease in the ratio of 1:30 and were incubated at 37 °C for 16 h. 1–2 μL of concentrated TFA (Trifluoro Acetic acid) was added after the incubation period was over to stop the reaction. The digested peptides were acidified by dissolving in 20ul of 0.1% formic acid before desalting.

The desalting was performed with C 18 membrane tips. The samples were again subjected to drying in Speed Vacc (Thermo Fischer Scientific) and dissolved in 20 μl of 0.1% FA (Formic Acid) in water [12]. The concentration of peptide determined using Bradford and subjected to ORBI TRAP analysis.