In-Solution Sample Digestion Protocol

Reagents Required

1 Ammonium bicarbonate (Sigma A6141) working concentration 50 mM
2 Dithiothreitol (Sigma D5545) working concentration 100 mM
3 Iodoacetamide (Sigma I1149) working concentration 200 mM
4 MS grade Trypsin (Sigma T6567) working concentration 0.4μg /μl
5 Formic acid (Merck 5.33002.0050)

Reagent Setup (prepare freshly before use)

  1. DTT solution: 100 mM DTT in 50 mM ammonium bicarbonate.
  2. Iodoacetamide solution: 200 mM Iodoacetamide solution in 50 mM ammonium bicarbonate.
  3. Formic acid 1% in milliQ water (v/v).
  4. Trypsin buffer: 0.4 μg /μl trypsin in 50 mM ammonium bicarbonate.

Trypsin digestion protocol for 100 μg of protein sample

  1. Add 5μl of 100 mM DTT solution to the protein sample.
  2. Incubate at 60° Celsius for 30 minutes
  3. Add 5 μl of 200 mM Iodoacetamide solution to the protein sample.
  4. Incubate at room temperature in dark for 30 minutes.
  5. Add trypsin buffer in the ratio of 1:25 (1μg of trypsin to 25μg of protein).
  6. Incubate at 37° Celsius overnight with gentle shaking.
  7. To arrest the reaction, add 1 μl of 1% formic acid (v/v) to the samples.
  8. Incubate at 37° Celsius for 20 minutes.
  9. Store the samples at -20 deep freezer until use.
In-Solution Sample Digestion Protocol