PROTOCOL
IN-SOLUTION DIGESTION OF PROTEINS

This protocol can be used to digest proteins prior to Mass Spectrometry Analysis.

Procedure:

  1. In general, proteins require denaturation and disulfide bond cleavage before enzymatic digestion can go to completion.
  2. Dissolve 1–10mg of the target protein in 8M urea, 50mM Tris-HCl (pH 8), 4mM DTT in a reaction volume of up to 1ml (25µl minimum). If your sample does not require denaturation (i.e. cell lysate, immunoprecipitations), then re-suspend your sample in 50-100µL of 50mM NH4HCO3 (pH=8.3).
  3. Add DTT to final volume of 10 mM.
  4. Heat at 60°C for 30 minutes.
  5. Allow sample to return to room temperature.
  6. Add iodoacetamide (made fresh in ddH2O) to a final volume of 10mM.
  7. Incubate at room temperature in the dark for 15 minutes.
  8. Inactivate any remaining iodoacetamide by adding DTT to a final concentration of 40mM.
  9. If 8M urea buffer was used, dilute out with 50mM NH4HCO3 until concentration is <1M urea.
  10. Check the pH of your solution – it should be around 7.5-8.5.
  11. Add modified trypsin (MS grade) to a final protease:protein ratio of 1:50-1:100 (w/w).
  12. Digest samples O/N @ 37°C.
  13. Lyophilize samples, or freeze and drop off at the SPARC facility.

 

 

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