Procedure for Tryptic Digest of Silver Stained Gels

  1. When using silver staining, the staining reagents and procedure must 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 (Pierce Silver Stain Kit for Mass Spectrometry; In particular, do not use BioRad silver stain!!) 
  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 ice cold 10 mM NH4HCO3 to all samples and the blank.
  5. Let stand for 90 min on ice to allow enzyme/buffer solution to absorb into the gel while keeping autolysis to a minimum.
  6. Add an additional 20 µl 10 mM NH4HCO3 that does not contain enzyme.
  7. Incubate at 37°C for 16-24 hours. If the caps are exposed to air, water will condense on the underside.  This could cause the gel pieces to dry out overnight.  Hence, you may want to carefully seal the tubes and submerge the caps.  If MALDI-MS is needed, samples may be submitted after this step.
  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 8 and 9, twice and
  11. Combine extracts with the supernatant from step 8.  Best practice is freeze at -80 > 3hr and then lyophilize.  (You should see no powder[detergent] or salt).
  12. Submit sample for MALDI-TOF MS and/or LC-Orbitrap MS/MS analysis
Hints For In Gel Digest Procedure
  • Always wear gloves when handling the gel.
  • Avoid dust, which contains sheep and human keratin. 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 tubes made for low protein binding.  We recommend Eppendorf or Axygen.
  • Use only sequencing grade trypsin. This may be obtained from Promega, Pierce, or Wako.
  • Due to concerns with reagent quality, lipid/detergent/etc. removal and sample concentration, we prefer to perform the digestions in the PCF.
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