In-Gel digestion


Use the In-Gel Tryptic Digestion Kit from Pierce, ThermoFisher or follow the protocol below


Trypsin in-gel digestion of proteins (pdf) for collodial coomassie stained polyacrylamide gel slices

Materials
All solvents should be HPLC grade, NEVER use pipette tips when transferring acids >2% in concentration!
Avoid Contaminations (pdf)

  1. Dithiothreitol DTT (Fisher, part # PI-20291); Stock solution: 1 M in H2O
  2. Iodoacetamide IAA (Fisher, part # AC12227-0050); Stock solution: 100 mM in H2O (0.0185g/ml; always prepare fresh, light sensitive)
  3. Urea (Fisher, part # AC14075-0010 )
  4. Water (Fisher, part # W6-4 optima LCMS grade)
  5. Ammonium bicarbonate (Fisher, part # A643-500) Stock solution: 500 mM in H2O (NH4HCO3 (79.1g/mol): 3.955g/100ml)
  6. Acetonitrile (Fisher, part # A955-4 optima LCMS grade)
  7. 1 μg/μL Trypsin in 0.01% acetic acid (modified, sequencing grade, Promega, part # V5111, 5 x 20μg/μg)
  8. Gel-Loading Pipet Tips (VWR, part # 53509-015)
  9. Eppendorf LoBind Microcentrifuge Tubes: Protein (Fisher, part # 13-698-794)

Avoid Keratin contamination

When handling the gel and gel band(s) use extreme caution to avoid keratin contamination (from your skin and hair etc.). Wear gloves at all times and use clean equipment (wipe all surfaces and tools with methanol/water and if possible use new unused razor blades). Keep the gel at a reasonable distance from your body, don't talk, sneeze etc over the gel.....

Avoid contamination from plasticizers

To avoid contamination from plasticizers, use all fresh solvents. Never use any plastic pipettes to transfer solvents from the original bottles, instead poor the solvent into a beaker. Repeated exposure of solvents to plastics will contaminate the stock solvents with plasticizers. Never use plastic pipettes when handling concentrated (>2%) acids, use glass pipettes or Hamilton syringes (rinse the syringe with water after using it for concentrated acids to avoid corrosion of the metal needle). If you don't know whether your stock solvents already are contaminated, buy new solvents and make sure no one is contaminating them! Keep your solvents in glass bottles, preferably with Teflon lined lids. Never use bottle lids that have foil backed cardboard liners!

Method

Use coomassie stained polyacrylamide gels of 1mm thickness.
Note: The excised band can be stored in de-ionized water for a few days at 4oC if necessary. Protein amounts should be in the hundreds of femtomole to 1 pmol range. A band that can be visualized by Coomassie blue stain, usually contains sufficient protein for identification.

This method involves reduction and alkylation of cysteine-containing peptides, this is useful for low level protein <1pmol and typically results in higher sequence coverage of the protein, as it improves the recovery of cystinecontaining peptides and minimizes the appearance of unknown masses in MS analysis from disulfide bond formation and side chain modification
Note: Alkylation with iodoacetamide increases the mass of a peptide by 57.02 for each cystine present. Acrylamide modification of cystine results in a peptide mass increase of 71.04.

For protein bands stained with mass spectrometry-compatible silver stains (e.g. Pierce Silver Stain Kit for Mass Spectrometry (24600)) or reversible zinc staining (Pierce Zinc Reversible Stain Kit (24582)), alternative destaining procedures will be required.

  1. Excise protein spot/band, cut into small pieces (~1 mm3) and dehydrate in acetonitrile for approx. 10 min, repeat this step twice. Remove acetonitrile and SpeedVac until dry. Note use gel loading pipette tips to remove solvents, and be careful not to lose the gel pieces.
  2. Add 50-100 μL 10mM DTT in H2O (or in 25mM ammonium bicarbonate) just enough to cover the gel pieces, vortex and spin down
  3. Incubate at 56 oC for 45 min to 1hr.
  4. Spin down, and pull off supernatant, allow to cool to room temperature.
  5. Alkylate with iodoacetamide (184.96g/mol: 18.5mg/ml)) 100mM in 25mM ammonium bicarbonate (5 to 10 fold excess over DTT), incubate for 30min in the dark at room temperature.
  6. Spind down, pull off supernatant, wash with H2O (or 25mM ammonium bicarbonate) and pull off wash.
  7. Dehydrate in acetonitrile for approx. 10min. Remove acetonitrile and SpeedVac untill dry.
  8. Rehydrate gel pieces at 4 oC for 45 min in buffer containing trypsin and 50 mM ammonium bicarbonate. (Approx. 5 μL/mm2 gel). The gel pieces should just be covered:
    Suggested amount of trypsin is 12.5 ng/uL of buffer for proteins that have been silver stained.
    (1μg/μL trypsin solution === 1μL/80μL 50 mM ammonium bicarbonate)
    Don't use more than 1 μg trypsin per sample for MS analysis.
  9. Cover gel pieces with 50 mM ammonium bicarbonate. Digest overnight at 37 oC (or at least for 3 hrs).
  10. Centrifuge gel pieces (4min) and collect (keep) supernatant. Use gel loading pipette tips to remove solvents, and be careful not to transfer the gel pieces as they could interfere with downstream MS analysis.
  11. Further extract peptides by one change of H2O and three changes of 5% formic acid in 50% acetonitrile incubate 20 min for each of the changes, centrifuge then collect at room temp.
  12. Reduce sample volume in speedvac to about 5μL, don't let the sample dry completely. Store at -20 oC
  13. Prior to LC-MS analysis add 0.1-0.2% formic acid in water to about 10-12 μL and inject 4-8 μL
Do not discard extracted gel pieces: if, for any reason, the digestion failed, it can be repeated with the same gel pieces using the same enzyme (trypsin) or using another enzyme. Note that, in the latter case, strong peptide background may be encountered because of the digestion of residual intact trypsin.

Resources

References

  1. In-gel digestion for mass spectrometric characterization of proteins and proteomes. Andrej Shevchenko, Henrik Tomas, Jan Havlis breve, Jesper V Olsen and Matthias MannNat Protoc. 2006;1(6):2856-60. link
  2. Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. Shevchenko A1, Wilm M, Vorm O, Mann M.Anal Chem. 1996 Mar 1;68(5):850-8. link