Trypsin lyc

The previously depleted proteins were subjected to precipitation using 5: 1 v/v cold acetone 100% v/v and incubated overnight at -20°C, then they were centrifuged at 15,000 g for 10 min, the supernatant was discarded, and the pellet was washed 3 times with acetone at 90% v/v, later the proteins were dried in a rotary concentrator at 4°C, and finally they were resuspended in 8 M urea with 25 mM of ammonium bicarbonate pH 8. The proteins were reduced using a final concentration of 20 mM DTT for 1 h, then they were alkylated incubating for 1 h with 20 mM iodoacetamide in the dark, then the proteins were quantified using the Qubit protein quantification kit. 10 ug of total proteins were diluted to 1 M urea using 25 mM ammonium bicarbonate pH 8, then the proteins were digested with trypsin/LyC (Promega) in a 1:50 ratio overnight at 37°C. The peptides were cleaned using Pierce C-18 Spin Columns (Thermo Scientific) using the protocol suggested by the manufacturer, the eluted peptides were dried using a rotary concentrator at 4°C and resuspended in 2% ACN with 0.1% v/v Formic Acid (MERCK), and quantified using Direct detect (MERCK Millipore).

The previously depleted proteins were subjected to precipitation using 5:1 v/v cold acetone 100% v/v and incubated overnight at −20°C, then they were centrifuged at 15,000 × g for 10 min, the supernatant was discarded and the pellet was washed three times with acetone at 90% v/v, later the proteins were dried in a rotary concentrator at 4°C, and finally they were resuspended in 8 M urea with 25 mM of ammonium bicarbonate pH 8.0.
The proteins were reduced using a final concentration of 20 mM DTT for one hour, then they were alkylated incubating for 1 h with 20 mM iodoacetamide in the dark, then the proteins were quantified using the Qubit protein quantification kit and 10 μg of proteins. The total was diluted to 1 M urea using 25 mM ammonium bicarbonate pH 8.0, then the proteins were digested with trypsin/LyC (Promega) in a 1:50 ratio overnight at 37°C. The peptides were cleaned using SepPack Vac C18 (Waters, USA) using the protocol suggested by the manufacturer, the eluted peptides were dried using a rotary concentrator at 4°C and resuspended in 2% ACN with 0.1% v/v formic acid (MERCK, Germany), and quantified using Direct detect (MERCK Millipore).