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Trypsin Digestion Protocols

Trypsin Digestion of Proteins in Solution (Adapted from Promega)

Trypsin Gold, Mass Spectrometry Grade (Promega, 100μg, cat.# V5280)

Storage Conditions: Store the lyophilized powder at –20°C. Reconstitute the powder in 50mM acetic acid and store at –20°C for up to one month. For long-term storage, freeze reconstituted trypsin at –70°C. Thaw the reconstituted trypsin at room temperature, placing on ice immediately after thawing. Remove the amount of trypsin needed, then refreeze the unused portion. To maintain maximum product activity, limit the number of freeze-thaw cycles to 5.

In general, proteins require denaturation and disulfide bond cleavage for enzymatic digestion to reach completion. If digestion of a native protein is desired, begin this protocol at Step 3.

  1. Dissolve the target protein in 6M guanidine HCl (or 8M urea), 50mM Tris-HCl (pH 8), 2–5mM DTT.
  2. Heat at 37°C for 45–60 minutes.
  3. For denatured proteins, add 50mM NH4HCO3 or 50mM Tris-HCl (pH 7.8), 1mM CaCl2, until the guanidine HCl or urea concentration is less than 1M. For digestion of native proteins, dissolve the protein in 50mM NH4HCO3 or Tris-HCl buffer with a pH between 7 and 9.
  4. Add Trypsin Gold to a final protease: protein ratio of 1:20 (w/w); it is desirable that protein concentration is at least 0.1 mg/ml. Incubate at 37°C for at least 4 hour to overnight. Remove an aliquot to determine the extent of digestion by subjecting the aliquot to reverse phase HPLC or SDS-PAGE. If further proteolysis is required, continue incubation at 37°C until the desired digestion is obtained (fresh trypsin can be added if necessary).
  5. The trypsin digestion can be stopped by freezing or by lowering the pH of the reaction below pH 4 by adding formic, acetic, or trifluoroacetic acid (trypsin will regain activity when the pH is raised above pH 4). Digested samples can be stored at -20°C.

In-Gel Trypsin Digestion of Proteins

In-gel protein digestion can be performed using commercially available kits from Promega or Pierce according to the manufacturers' protocols. See the Pierce protocol.