Target Discovery and Validation Reviews and Protocols

80 Beaty et al.
6. Shake the gel for at least 7 h or until the background becomes clear. The water
should be changed several times during destaining (see Note 25).
3.3.7. In-Gel Tryptic Digestion of Proteins Resolved
by 1D Gel Electrophoresis
After visualization of the proteins by either silver staining or colloidal
Coomassie staining, the resolved proteins are digested by trypsin before mass
spectrometry analysis (17).
1. Excise the gel lane into approx 20–30 bands (depending on the size of the gel).
2. Cut each band into smaller pieces (~2 × 2 mm).
3. Wash the gel pieces from each band with Milli-Q water and water:acetonitrile 1:1
(v/v) two times for 15 min at room temperature. Solvent volumes in the washing
step roughly equaled five times the gel volume.
4. After washing, remove the liquid and cover gel pieces in 100% acetonitrile to
shrink the gel pieces.
5. After approx 5 min, remove acetonitrile and incubate the gel pieces it with 10 mM
DDT in 100 mM ammonium bicarbonate (NH 4 HCO 3 ) for 45 min at 56°C to
reduce the cysteines.
6. Subsequently, remove the liquid and replace by 55 mM iodoacetamide in 100 mM
NH 4 HCO 3 and then incubate for 30 min at room temperature in the dark to
alkylate the cysteines.
7. Remove the iodoacetamide solution surplus and wash the gel pieces two times in
water and acetonitrile as described above and subsequently dehydrate by adding
100% acetonitrile.
8. Rehydrate the gel pieces by incubation in a digestion buffer containing
12.5 ng/µL trypsin (modified sequencing grade, Promega) in 50 mM NH 4 HCO 3
and incubate for 45 min on ice. Enough digestion buffer is added to cover the
gel pieces.
9. After 45 min, replace the digestion buffer by approximately the same volume
50 mM NH 4 HCO 3 but without trypsin to keep the gel pieces wet during enzymatic
digestion.
10. Incubate the samples at 37°C overnight.
11. After digestion, remove the remaining supernatant and save it in a 1.5-mL tube.
12. Extract the remaining peptides from the gel pieces by incubating in 5% formic
acid (enough to cover the pieces) for 15 min and then adding the same volume of
100% acetonitrile for further 15-min incubation.
13. Repeat the extraction twice. Collect all three fractions in one tube and dry down
in a vacuum centrifuge.
14. Resuspend in 10–20 µL of 5% formic acid.
3.3.8. Liquid Chromatography-Tandem Mass Spectrometry Analysis
Depending on the type of sample analyzed and available hardware (e.g.,
HPLC system and mass spectrometer), the LC-MS/MS setup can be modified