In-gel Tryptic Digest for Protein ID by MS¶
Contributed by Timothy Mitchison, Harvard Medical School, Boston, MA, United States
In-gel Tryptic Digest for Protein ID by Mass Spectrometry (David Miyamoto, 2/12/2002). This protocol is based on Shevchenko A, Wilm M, Vorm O, & Mann M. Mass spectrometric sequencing of proteins from silver-stained polyacrylamide gels. Anal Chem 1996, 68:850-8. I have used it with success on both Coomassie and silver-stained gel bands.
The procedure includes reduction and acetamidation steps that may be skipped if desired. For heavily stained Coomassie bands, it is helpful to wash gel pieces for 1 hr in 100 mM NH4HCO3 prior to dehydrating with acetonitrile (step 2).
- Excise band from Coomassie or silver stained gel. Cut gel band into 1 mm cubes using clean razor blade on a clean glass surface. Transfer to an Eppendorf tube.
- Remove excess water with pipet. Add 25-35 µL acetonitrile to tube to cover gel pieces. Incubate 10 minutes at RT to dehydrate and shrink gel pieces.
- Remove acetonitrile with pipet. Speed-vac to dryness for 10 minutes.
- Swell gel particles in 150 µL 10 mM DTT in 100 mM NH4HCO3. Incubate 1 hour at 56°C.
- Cool to RT. Replace DTT solution with 150 µL 55 mM iodoacetamide in 100 mM NH4HCO3. Incubate 45 minutes at RT in the dark with occasional vortexing.
- Remove solution and wash gel pieces with 150 µL 100 mM NH4HCO3. Incubate 10 minutes at RT.
- Remove NH4HCO3 solution with pipet. Add 150 µL acetonitrile to dehydrate gel pieces. Incubate 10 minutes at RT.
- Repeat wash steps 6 through 7. Remove acetonitrile and speed-vac to dryness for 10 minutes.
- Place tubes in ice water bath and swell gel particles in 25-35 µL digestion buffer. Incubate 45 minutes in ice water bath. Digestion buffer consists of 12.5 ng/µL trypsin (Promega sequence-grade modified porcine trypsin, Cat. #V511A) in 50 mM NH4HCO3. To make the digestion buffer, dissolve 20 µg Promega trypsin in 80 µL Promega trypsin buffer solution (50 mM acetic acid), and dilute with 50 mM NH4HCO3 to 12.5 ng/µL.
- Remove trypsin-containing buffer. Add 5-10 µL 50 mM NH4HCO3 without trypsin to keep pieces wet during cleavage. Incubate o/n at 37°C.
- Spin 1’ at 14,000 rpm to spin down gel pieces. Save supernatent in a separate PCR tube.
- Add 20 µL 20 mM NH4HCO3 to cover gel pieces. Incubate 10 minutes at RT. Transfer supernatent to the PCR tube from step 11.
- Add 25 µL 5% formic acid, 50% acetonitrile to the gel pieces. Incubate 20 minutes at RT.
- Spin 1’ at 14,000 rpm. Remove formic acid/acenonitrile solution and save in the same PCR tube from step 11.
- Repeat formic acid extraction (steps 13 through 14) twice more.
- Dry PCR tube in speed-vac to complete dryness. Store at -20°C until analysis.
Shevchenko A, Wilm M, Vorm O, Mann M Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. Anal Chem (1996) pmid:8779443