Thesis final - after defense-7 - Jacobs University

Chapter 2
Table 2: The in-gel digestion protocol optimized specifically for the proteins fractionated by
HIC
Steps
Description
1. Washing the gels The gel pieces were washed overnight, with 50% (v/v) methanol
and 5% (v/v) acetic acids for the removal of salts and other
impurities.
2. De-staining After washing, the gel pieces were incubated in 200 µl of 100
mM ammonium carbonate/acetonitrile (1:1 v/v) for 30 minutes
to destain the gels.
3. Reduction After destaining, 50 µl of 10 mM DDT dissolved in 100 mM
ammonium carbonate were added to gel pieces and incubated
for 45 minutes at 56°C to reduce the disulfide bridges.
4. Alkylation Then 100 µl of 55 mM iodoacetamide was added to samples and
incubated for 20 minutes in the dark to inhibit the reformation of
disulfide bonds and alkylate the free cysteins.
5. Trypsin digestion Then 10 µl of trypsin and 90 µl of 25 mM ammonium
bicarbonate in 9% acetonitrile solution were added to each
sample and incubated in ice for 3 hours to absorb maximum
enzymes. The samples were then incubated for 16 hours at 37°C
in the digestion buffer for protein digestion.
6. Extraction of
peptides
7. Concentration of
the peptide extract
a. 200 µl of Milli Q water was added to each sample and
incubated for 15 minutes at 37°C to extract hydrophilic
peptides.
b. 200 µl of extraction buffer (1:2 v/v 5% formic
acid/acetonitrile) was added to each sample and
incubated for 15 minutes at 37°C to extract hydrophobic
peptides.
The peptide extract was concentrated and 15 µl of 0.5% v/v
TFA was added to each sample, followed by centrifugation at
10,000 rpm for 10 minutes. The samples were then ready for
identification by MALDI-ToF-MS.
After digestion, the sample was collected as supernatant and 3 µl of the sample was spotted
on the MALDI target plate followed by 0.5% TFA for washing. The sample was air dried at
room temperature. All mass spectra were calibrated using peptides from the auto digestion of
trypsin. The peak lists were searched using the Mascot search engine
30