Proteomics Facility

  • Tryptic Digest Procedure for Silver Stained Gels

    1. When using silver staining, the Tryptic Digest technique needs to be modified in order to successfully use MALDI-TOF MS for protein identification (Shevchenko, A., et al., 1996). There are commercially available silver staining kits that are designed for MS compatibility (SilverQuest Kit from Invitrogen and Proteosilver Plus Stain Kit from Sigma-Aldrich).
    2. Speedvac gel pieces to COMPLETE dryness
    3. For reduction and alkylation, see Procedure for Reduction and Alkylation
    4. Add 0.1 µg modified trypsin per 15 mm3 of gel in 15 µl 10 mM NH4HCO3 to all samples and the blank
    5. Let stand for 5-10 minutes to allow enzyme/buffer solution to absorb into the gel
    6. Add an additional 20 µl 10 mM NH4HCO3 that does not contain enzyme
    7. Incubate at 37° C for 16-24 hours
    8. Remove supernatant containing peptides and add to a 1.5 ml Eppendorf prewashed (see step 1 ) tube.
    9. Extract the gel by adding 50 µl 0.1% TFA, 60% CH3CN and shaking at room temperature for 60 minutes (use more volume if necessary)
    10. Repeat steps 19 and 20
    11. Combine extracts with the supernatant from step 18
    12. Submit sample for MALDI-TOF MS and 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.