Proteomics Facility

  • Procedure for In Gel Tryptic Digest and Extraction for Coomassie-stained gels for MALDI-ToF MS and NanoLC LTQ MS/MS

    1. Prewash 500 µl Eppendorf tubes with 500 µl 0.1% FA/60% CH3CN.
    2. Put the Coomassie Blue stained gel band into one prewashed tube.
    3. Also take a blank section of gel that should not contain protein and put in a prewashed 500 µl tube.
    4. Add250 µl 50% H2O/50% acetonitrile and wash for 5 minutes.
    5. Remove wash.
    6. Add 250 µl 50% CH3CN/ 100 mM NH4HCO3 to all samples. (*Concentration of NH4HCO3 may depend on the amount of protein in bands.)
    7. Cut gel bands into smaller pieces using a pair of tweezers.
    8. Wash for 30 minutes at room temperature.
    9. Remove wash being sure not to remove gel pieces.
    10. Add 250 µl 50% CH3CN/10 mM NH4HCO3 to the gel pieces.
    11. Wash for 30 minutes at room temperature.
    12. Remove wash.
    13. Turn on water bath to 56°C.
    14. Speedvac gel pieces to COMPLETE dryness.
    15. Add enough 10 mM DTT solution (~50 µl) to cover the gel pieces, reduce for 1 hr at 56°C. After this step, the water bath should be cooled to 37°C for trypsin digestion (replace some warm water with room temperature water).
    16. Cool samples to room temperature and replace the DTT solution with roughly the same volume of 55 mM iodoacetamidesolution. Incubate for 45 min at room temperature in the dark with occasional vortexing.
    17. Wash gel pieces (rehydrate) with ~100 µl of 25 mM ammonium bicarbonate, pH 8, for 10 min while vortexing, and dehydrate with ~100 µl of 25 mM ammonium bicarbonate/50% acetonitrile.
    18. Repeat rehydration and dehydration.
    19. Remove the liquid phase and dry gel pieces to completion in vacuum centrifuge.
    20. Addappropriate trypsin concentration in 25 mM NH4HCO3to all samples and the blank. For strong bands, 10 ng/µl trypsin is recommended. For light bands, 8 ng/µl trypsin is recommended.
    21. Let stand for 5-10 minutes to allow enzyme/buffer solution to absorb into thegel.
    22. Add an additional 10-20 µl 25 mM NH4HCO3 that does not contain enzyme.
    23. Incubate at 37°C for 16-24 hours. If MALDI-MS is needed, samples may be submitted after this step.
    24. Remove supernatant containing peptides and add to a 500 µl Eppendorf prewashed (i.e., see step 1) tubes.
    25. Extract the gel by adding 50 µl 0.1% FA, 50% CH3CN (use more volume if necessary).
    26. Sonicate samples at room temperature for 10 minutes.
    27. Shake samples at room temperature for 60 min.
    28. Centrifuge samples at 1100 xg for 6.5 minutes.
    29. Remove supernatant (being careful not to remove gel pieces) and combine with supernatant in step 24.
    30. Repeat steps 25-29 two more times for a total of three extractions.
    31. Speedvac combined supernatant until there is approximately 5 µl left in tube. Submit sample for LTQ MS/MS analysis.

    Hints For In Gel Digest Procedure

    • Always wear gloves when handling the gel
    • Avoid dust, which contains sheep and human keratins. Wipe down work surfaces, tubes and scalpel with lint-free cloth moistened with methanol/water solution
    • Excise the band from the gel with a sharp scalpel in such a manner as to avoid removing excess gel
    • Place the excised band in an Eppendorf tube (that does not contain a rubber O-ring) and freeze. Do not leave the gel in destain buffer or in any other liquid. Also, it is not necessary to dry the gel band.
    • Excise a similar size piece of "blank" gel (that does not contain any protein) and put it in a separate Eppendorf tube so that this blank section of gel can be used as a control to identify artifact peaks.
    • Cut the gel bands into pieces that are ~1-2 mm3 in size
    • Solvents should be the highest purity. Use distilled and deionized water.
    • Use siliconized tubes to reduce contact with plastics.
    • Use only sequencing grade trypsin. This may be obtained from Promega or Sigma.
    • For reasons of reagent quality and sample concentration, we prefer investigators submit the sample after finishing step 18.

    References

    Shechenko, A.; Wilm, M.; Vorm, O.; Mann, M. Mass spectrometric sequencing of proteins from silver-stained polyacrylamide gels. Electrophoresis, 1996, 68, 850-858.

    Jimenez, C.R., Huang, L., Qiu, Y., and Burlingame, A.L. Current Protocols in Protein Science, John Wiley and Sons: New York, 1998; pp 16.4.1-16.4.5

    Kinter, M.; Sherman, N.E. Protein Sequencing and Identification Using Tandem Mass Spectrometry; Wiley-Interscience: New York, 2000; Chapter 6.