Keck Home Page > Protein Chemistry > Gel Tryptic Digest Procedure

Procedure for In Gel Tryptic Digest

1. Prewash 3 x 1.5 ml Eppendorf tubes with 500 µl 0.1% TFA/60% CH₃CN
2. Put the Coomassie Blue stained gel band, which should be cut into small pieces, into one prewashed tube
3. Remove one lane of a transferrin control (supplied by Keck Facility) containing 25 pmol and put in a separate prewashed 1.5 ml tube
4. Also take a blank section of gel (supplied by investigator-user) that should not contain protein and put in a prewashed 1.5 ml tube
5. Add 250 µl 50% H₂O/50% acetonitrile and wash for 5 minutes
6. Remove wash
7. Add 250 µl 50% CH₃CN/ 50 mM NH₄HCO₃ to all samples
8. Wash for 30 minutes at room temp on tilt table
9. Remove wash
10. Add 250 µl 50% CH₃CN/10 mM NH₄HCO₃ to the gel pieces
11. Wash for 30 minutes at room temperature on a tilt table
12. Remove wash
13. Speedvac gel pieces to COMPLETE dryness
14. Add 0.1 µg modified trypsin (Promega) per 15 mm³ of gel in 15 µl 10 mM NH₄HCO₃ to all samples and the blank
15. Let stand for 5-10 minutes to allow enzyme/buffer solution to absorb into the gel
16. Add an additional 20 µl 10 mM NH₄HCO₃ that does not contain enzyme
17. Incubate at 37C for 24 hours
18. Hold all samples in the refrigerator pending MALDI-MS protein identification
19. For MALDI-MS take 1 µl of each supernate and mix with 1 µl of internal standards (containing 100 fmol each of two standards) and 1 µl -cyano matrix (4.5 mg/ml) and then subject to MALDI-MS for peptide mass searching. Store the remaining gel in the refrigerator while the MALDI-MS is in progress
20. If peptide mass searching does not identify the sample, extract the peptides by adding 200 µl 0.1% TFA, 60% CH₃CN and shaking at room temperature for 60 min (use more volume if necessary)
21. Remove wash containing peptides and add to a 1.5 ml Eppendorf prewashed (i.e., see step 1) tube
22. Repeat step 20 and 21
23. Speedvac dry the combined washes
24. Samples destined for LC/MS/MS protein identification on the LCQ should be dissolved in 60 µl 0.05% TFA, 5% acetonitrile while samples destined for preparative HPLC should be dissolved in 45 µl of the same solvent
25. Filter all samples - for LC/MS/MC go to step 26, for preparative HPLC go to step 28
26. Load 14 µl of the filtered extract onto the LC/LCQ mass spectrometer which will then inject 10/60 µl or 16.7% of the original digest
27. To collect the remaining digest (assuming that 16.7% had been subjected to LC/LCQ mass spectrometry and had screened and not been identified), transfer the remaining 46 µl of the digest into the HP1090 sample vial, rinse the Eppendorf tube that contained the digest with 10 µl 100% TFA and add to the HP1090 sample vial and vortex. The HP1090 will then inject 50 µl or 50/56 x 46/60 = 68% of the original digest
28. To collect the remaining digest (assuming that no LC/LCQ analysis was carried out) transfer the remaining 45 µl of the digest into the HP1090 sample vial, rinse the eppendorf tube that contained the digest with 10 µl 100% TFA and add to the HP1090 sample vial and vortex. The HP1090 will then inject 50 µl or 50/55 = 91% of the original digest - not taking into account the MALDI screen
29. Carry out reverse phase HPLC using a Vydac C18 1 x 250 mm column run at 30 µl/min (More information on preparative HPLC)

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Last modified: 23-Oct-2006 (GB)