In-gel Tryptic Digest for Protein ID by Mass Spectrometry

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).

1.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.

2.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.

3.Remove acetonitrile with pipet.Speed-vac to dryness for 10 minutes.

4.Swell gel particles in 150 μl 10 mM DTT in 100 mM NH₄HCO3.Incubate 1 hr at 56℃.

5.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.

6.Remove solution and wash gel pieces with 150 μl 100 mM NH₄HCO₃.Incubate 10 minutes at RT.

7.Remove NH₄HCO₃ solution with pipet.Add 150 μl acetonitrile to dehydrate gel pieces.Incubate 10 minutes at RT.

8.Repeat wash steps 6 through 7.Remove acetonitrile and speed-vac to dryness for 10 minutes.

9.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.

10.Remove trypsin-containing buffer.Add5-10 μl 50 mM NH₄HCO₃ without trypsin to keep pieces wet during cleavage.Incubate o/n at 37℃.

11.Spin 1' at 14,000 rpm to spin down gel pieces.Save supernatent in a separate PCR tube.

12.Add 20 μl 20 mM NH₄HCO₃ to cover gel pieces.Incubate 10 minutes at RT.Transfer supernatent to the PCR tube from step 11.

13.Add 25 μl 5% formic acid,50% acetonitrile to the gel pieces.Incubate 20 minutes at RT.

14.Spin 1' at 14,000 rpm.Remove formic acid/acenonitrile solution and save in the same PCR tube from step 11.

15.Repeat formic acid extraction (steps 13 through 14)twice more.

16.Dry PCR tube in speed-vac to complete dryness.Store at -20℃ until analysis.