Mix carefully and carry out the digestion overnight at 37 °C. 2. Perform a second in-solution trypsin digestion in 80% ACN. Add trypsin in a 1:100 ratio regarding the total protein content of your sample. Add the calculate volume of 100% acetonitrile in order to achieve 80% Acetonitrile in final sample volume and incubate at 37 °C for 3 hours.