In-gel Digestion of Silver Stained Proteins
(with DTT Reduction and Iodoacetaminde Alkylation)

1. Always wear gloves and work in the hood to reduce keratin contamination.

2. Excise gel bands or spots.

3. It is highly recommended that a positive and negative control be conducted in parallel to all analyses. The positive control can be a marker or known protein while the negative control should be a blank region of the gel.

4. Wash twice with 50µL of 18 Mohm water for approx 10 minutes each. (Washing steps 3 through 5 are included to remove possible surface contaminants arising during spot/band excision and to remove residual ionic and nonvolatile buffers)

5. Wash once with 50 µL of a 50%/50% solution of acetonitrile for 10 minutes.

6. Wash and dehydrate with 50 µl (spot) to 100 µL (band) of acetonitrile until gel plugs turn opaque, generally 20minutes. Substituion of two 10min acetonitrile washes is a faster option.

7. Decant all liquid from the opaque gel plugs.

8. Dry sample plugs in vacuum centrifuge.

9. Rehydrate and incubate samples in 10mM dithiothreitol (DTT) in 25mM ammonium bicarbonate at 56°C for 1 hour. Solution volume should be sufficient to cover gel plugs or bands, ie 20-50 µL (10mM DTT is approximately a 1.5mg/µL solution of DTT).

10. Allow samples to cool to room temperature.

11. Replace 10mM DTT solution with an equal volume of 55mM iodoacetamine in 25 mM ammonium bicarbonate and incubate in the dark for 45 minutes.

12. Decant iodoacetamide solution.

13. Wash with 25 mM ammonium bicarbonate solution for 10 min followed by a 10 min dehydration with 100% acetonitrile.

14. Rehydrate with 25 mM ammonium bicarbonate solution for 15 min. Decant 25 mM ammonium bicarbonate solution and dehydrate for 10 min with 100% acetonitrile.

15. Remove all liquid and dry sample plugs in vacuum centrifuge.

16. Prepare trypsin solution while drying samples using sequence grade, modified bovine or porcine Trypsin (Roche/Promega).
    Prepare stock trypsin solution by dissolving 20 to 25 µg in 200µL 1mM HCl or 1% Acetic acid so that final concentration is between 100ng/µL to 125 ng/µL

17. Prepare working trypsin solution by diluting stock solution 1 to 10 with 25 mM ammonium bicarbonate such that final concentration = 10 to 12.5 ng/µL.
    Note the pH difference in stock solution (~2.9) versus working solution (pH~8.0). Trypsin activity is pH dependent. At low pH, trypsin has low activity and can be stored for approximately 2weeks. Trypsin working solutions must be used shortly after increasing pH to 8, ie 2 hrs. If not autolytic digestion products are observed at higher levels than desirable.

18. Rehydrate dried gel plug/bands in approximately 20 µL trypsin for 20 minutes. This allows the trypsin to infuse into the gel plug for "in-gel" digestion.

19. Remove excess trypsin solution with pipette. (some use a 25mM ammonium bicarbonate washed to aid in excess trypsin removal but we suggest that you only wash if autolytic trypsin peaks are problematic)

20. Add 10 to 20 µL of 25mM ammonium bicarbonate to sample vial to ensure proper hydration during digestion at elevated temperatures.

21. Digest at 37°C for 4 to 6 hrs. Overnight digestions are possible but yield higher levels of autolytic trypsin products which may decrease sensitivity for low level protein digests.

22. Stop digestion by adding 25 µL of 10 % formic acid (note pH change) allow to stand for 15 minutes.

23. Recover supernate – one can sample this solution directly for peptide mass mapping or continue extracting additional peptides. We suggest further extraction.

24. Extract twice with of 50%/50% acetonitrile/50mM ammonium bicarbonate for 15 minutes to recover additional peptides. Pool with supernate from step 12.

25. Extract with 25 µL to 50 mL of 100% acetonitrile. Pool with supernate from step 12.

26. Concentrate to 5 µL or dryness in vacuum centrifuge. Concentration to dryness is more convenient but may result in some loss of peptides which will not resolubilize.

27. Dried peptides can be stored at -20°C or -80°C for long periods of time (1yr).

28. Immediately prior to mass analysis dissolve dried peptides in 50%/50% acetonitrile/1% formic acid. Alternatively 0.1% trifluoroacetic acid can be used but TFA can reduce ionization efficiency through ion pair formation.

Comments: great care should be taken not to touch gels, allow hairs to fall on gels, or allow wools to come into contact with gels prior to staining or digestion. All these can contribute protein contaminants which will appear and interfere with the final peptide mass map.

References:
Shevchenko, A., Wilm, M., Vorm, O., Mann, M., 1996, Mass Spectrometric Sequencing of Proteins from Silver-Stained Polyacryllamide Gels, Anal. Chem. 68, 850-858.