InGelDigestionofProteinsSeparatedbyPolyacrylamideGelElectrophoresis

1. Excision of protein bands (spots) from polyacrylamide gels

Rinse the gloves you use with water to avoid traces of dust in your sample.

• Rinse the gel with water.

• Excise spots with clean pipette tip (f 2 mm) cutting as close to the edge of the spot as possible (to reduce the volume of ''background'' gel)

• Transfer gel spot into a well of a 96-microtitre plate (Costar # 3363 + 3092)

2. Reduction and alkylation

This step is omitted because proteins are already reduced and alkylated during the equilibration after the 1st dimension. Include maybe for small protein amounts.

3. Washing of gel pieces (Coomassie, Sypro Ruby)

Washing to remove unpolymerized acrylamide (which may react with the peptides) and other (buffer) contaminants (which may give a higher background during MS measurement)

• Wash the sample 5 min in 80 µl 0.1M NH4HCO3

• Add an equal volume of 100% Acetonitrile and vortex for 5 min

• Spin down the gel particles (table-top centrifuge, max. 2800 rpm)

• Remove the solution

Repeat this washing step twice.

• Shrink the gel pieces with 150 µl 100% Acetonitrile (HPLC grade) for about 5 min

• Remove the solution

• Dry down gel particles in a Speed Vac

4. In-Gel digestion with Trypsin

• Precool tubes with the dried gel particles and the Trypsin solution on ice

• Rehydrate the gel particles in the trypsin solution (12.5 ng/µl Trypsin [Promega #V5111] in 50 mM NH4HCO3) for 30-45 minutes at 4°C. Add just enough solution (20 µl) to cover the gel pieces (you only need to have the trypsin in the gel pieces to digest the proteins)

• After 15 minutes, check out the samples and add more trypsin solution if the solution is completely absorbed by the gel pieces

• After a total of 30-45 min rehydration, remove the remaining trypsin solution containing unnecessary Trypsin (to reduce the amount of self-digested trypsin peptides in the final sample)

• Wash briefly with 80 µl 50 mM NH4HCO3 (just add and remove) to remove the trypsin on the outside of the gel pieces

• Add 80 µl 50 mM NH4CO3 without Trypsin to have the gel pieces covered during the overnight digestion (16 - 20 h) at 37°C

• Spin down condensed water droplets

• Transfer liquid supernatant (''digest-solution'') which can already contain small peptides (<2000Da) to a fresh 0.5 ml tube or a 96-tubes-plate

5. Extraction of peptides from the gel piece

• Add 10-15µl of 25 mM NH4HCO3 (maybe more) (to adjust the pH)

• Incubate for 15 min at 37°C while shaking (Vortex)

• Spin down and add 100% Acetonitrile (1-2 times equal the volume of the gel, 80 µl)

• Incubate for 15 min at 37°C while shaking

• Sonicate for 5 min in sonication bath at 37°C (17L )

• Spin down liquid and transfer the ''basic extracted'' peptide containing solution to the same tube as the ''digest solution''

• Add 40-50 µl of 5% formic acid (to adjust the pH)

• Incubate for 15 min at 37°C while shaking

• Spin down and add 100 % Acetonitrile (1-2 times equal the volume of the gel, 80µl)

• Incubate for 15 min at 37°C while shaking

• Sonicate for 5 min in sonication bath at 37°C

• Spin down and transfer the ''acid extracted'' peptide containing solution to the same tube, together with the basic extracted peptides and the ''digest solution''

• Freeze in liquid nitrogen, then dry in a Speed-vac.

At this point, samples can be stored at -20°C.

6. Mass spectrometry

• Dissolve the sample in ~ 10 µl 0.1% TFA

• Inject ~ 5 µl in the LCQ Deca from Finnigan

2. Reduction and alkylation

• Wash the particles with 100-150 µl of water (5min), spin down and remove the liquid.

• Add acetonitrile (3-4 gel volumes) and wait for 10-15 min, spin particles down and remove all liquid, dry down in a speed vac.

• Swell the gel pieces in 10 mM DTE/ 0.1 M NH4HCO3, enough to cover the gel, and incubate for 30 min at 56°C (waterbath) to reduce the protein.

• Spin down and remove excess liquid, shrink with acetonitrile.

• Replace acetonitrile with 55 mM Iodoacetamide/ 0.1 M NH4HCO3, incubate for 20 min at room temperature in the dark.

• Remove iodoacetamide solution, wash the gel particles with 150-200 µl 0.1 M NH4HCO3 for 15 min.

• Spin down, remove all liquid and shrink with acetonitrile.

• Remove all liquid and dry gel particles down in a speed vac.