3.3 Trypsin Digestion
of Protein Complexes
and Preparation
for Mass Spectrometry
Analysis
3.3.1 Eluted Sample
Preparation
This method is an universal method of sample preparation and
digestion that works with all samples eluted in LDS NuPAGE
buffer and has the main advantage to remove all small putative
contaminants as detergents, small peptides, and salts that could
interfere with protease activities or MS analysis. The methods consist of:
1. Load 90% of the remaining sample on a 4–12% Bis/Tris gel
2. Stop the electrophoresis migration as soon as the sample has
completely entered the top of the gel (80 V, 5 min).
3. Stain the acrylamide gel overnight with the Imperial protein
stain, according to the manufacturer instructions.
4. The next day, wash the gel extensively with water until a clean
stacking band appears.
5. Stacking bands highlighted with trans illuminator are cut from
gel using a clean scalpel and transferred onto a 96-well polypropylene plate with conical bottoms and stored drained until
reduction–alkylation–digestion steps (see Note 22).
3.3.2 Trypsin Digestion
Once cut, the sample is directly processed for trypsin digestion.
1. In each well, wash the gel pieces with 100 μL of washing buffer
1, incubate for 5 min at room temperature and aspirate the
extra liquid. Repeat once.
2. Add 100 μL of CH3CN, the gel will compact and will become
white, then remove the liquid and let the gel dry for 15 min at
room temperature.
3. Add 50 μL of rehydration buffer and let the gel swell for 5 min.
4. Add 50 μL of CH3CN, wait 15 min and remove the liquid and
let the gel dry for 15 min (see Note 23).
5. The sample is reduced with the addition of 100 μL of reduction
buffer and heated to 56
C for 45 min. Cover the plate with an
adhesive film to avoid contaminations and liquid evaporation.
6. Remove the liquid and replace rapidly with fresh 100 μL of
alkylation buffer and incubate in the dark for 30 min at room
temperature.
7. Liquid is removed from the well, and gel is washed twice with
washing buffer 2. Then the liquid is removed and the gel is dry
off during 15 min at room temperature.
8. The sample is then digested by adding 50 μL of sequence-grade
trypsin prepared at 12.5 ng/μL in digestion buffer.
9. Incubate for 15 min at 4
C
10. Add 50 μL of digestion buffer to fully rehydrate gel and
incubate overnight at 37
C.
11. The day after, add 75 μL of 5% formic acid.
30
Avais M. Daulat et al.
of Protein Complexes
and Preparation
for Mass Spectrometry
Analysis
3.3.1 Eluted Sample
Preparation
This method is an universal method of sample preparation and
digestion that works with all samples eluted in LDS NuPAGE
buffer and has the main advantage to remove all small putative
contaminants as detergents, small peptides, and salts that could
interfere with protease activities or MS analysis. The methods consist of:
1. Load 90% of the remaining sample on a 4–12% Bis/Tris gel
2. Stop the electrophoresis migration as soon as the sample has
completely entered the top of the gel (80 V, 5 min).
3. Stain the acrylamide gel overnight with the Imperial protein
stain, according to the manufacturer instructions.
4. The next day, wash the gel extensively with water until a clean
stacking band appears.
5. Stacking bands highlighted with trans illuminator are cut from
gel using a clean scalpel and transferred onto a 96-well polypropylene plate with conical bottoms and stored drained until
reduction–alkylation–digestion steps (see Note 22).
3.3.2 Trypsin Digestion
Once cut, the sample is directly processed for trypsin digestion.
1. In each well, wash the gel pieces with 100 μL of washing buffer
1, incubate for 5 min at room temperature and aspirate the
extra liquid. Repeat once.
2. Add 100 μL of CH3CN, the gel will compact and will become
white, then remove the liquid and let the gel dry for 15 min at
room temperature.
3. Add 50 μL of rehydration buffer and let the gel swell for 5 min.
4. Add 50 μL of CH3CN, wait 15 min and remove the liquid and
let the gel dry for 15 min (see Note 23).
5. The sample is reduced with the addition of 100 μL of reduction
buffer and heated to 56
C for 45 min. Cover the plate with an
adhesive film to avoid contaminations and liquid evaporation.
6. Remove the liquid and replace rapidly with fresh 100 μL of
alkylation buffer and incubate in the dark for 30 min at room
temperature.
7. Liquid is removed from the well, and gel is washed twice with
washing buffer 2. Then the liquid is removed and the gel is dry
off during 15 min at room temperature.
8. The sample is then digested by adding 50 μL of sequence-grade
trypsin prepared at 12.5 ng/μL in digestion buffer.
9. Incubate for 15 min at 4
C
10. Add 50 μL of digestion buffer to fully rehydrate gel and
incubate overnight at 37
C.
11. The day after, add 75 μL of 5% formic acid.
30
Avais M. Daulat et al.