11. Add 0.2 M ammonium bicarbonate in 10–15 μL increments
until the final volume just covers the tops of the completely
swollen gel pieces (see Note 29).
12. Incubate the gel slices for 12–18 h at 37
C.
13. Add 100 μL of the extraction solution to the gel slices and
vortex.
14. Incubate the samples at 37
C for 20 min.
15. Vortex and incubate for an additional 20 min at 37
C.
16. Centrifuge the samples briefly and transfer the supernatant in a
new deplasticized microcentrifuge tube. Avoid pipetting any
gel slices into pipet tips when collecting supernatant.
17. Repeat steps 13–15, combining the supernatant in the tube
from step 15.
18. Place the combined supernatant extract in the vacuum concentrator to remove acetonitrile and concentrate the peptide
digest (see Note 30).
19. Resuspend in MS loading solvent so that sample is roughly at a
concentration of 0.5–1 μg/μL (see Note 31).
20. Centrifuge samples at 14,000 Â g for 5 min.
21. Gently pipet the sample out of the tube, being sure not to
touch the bottom of the tube with the pipet tip (there will be a
small volume left in the tube), and transfer into an autosampler
vial. Make sure not to introduce any bubbles into the vial. Cap
and label vials.
22. Samples can be stored at À20
C until further processing for
LC-MS analysis.
3.6 In-Solution
Digestion Processing
for Bottom
up Proteomics (See
Note 24)
1. Precipitate 30 μg of protein sample with four volumes of prechilled acetone for 1 h at À20
C (see Note 32).
2. Pellet the protein by centrifugation at 15,000 Â g for 10 min at
4
C.
3. Carefully discard the supernatant and rinse the pellet with
300 μL of À20
C acetone.
4. Pellet the protein by centrifugation at 15,000 Â g for 10 min at
4
C.
5. Carefully discard the supernatant and let the pellet dry for
$5 min.
6. Dissolve the protein pellet in 15 μL of 8 M urea.
7. Sonicate the sample in the bath sonicator for 5 min.
8. Add 20 μL of 0.2% ProteaseMax surfactant solution.
9. Vortex for 5–10 min.
10. Add 58.5 μL of 50 mM ammonium bicarbonate.
542
Carolina Mehaffy et al.
until the final volume just covers the tops of the completely
swollen gel pieces (see Note 29).
12. Incubate the gel slices for 12–18 h at 37
C.
13. Add 100 μL of the extraction solution to the gel slices and
vortex.
14. Incubate the samples at 37
C for 20 min.
15. Vortex and incubate for an additional 20 min at 37
C.
16. Centrifuge the samples briefly and transfer the supernatant in a
new deplasticized microcentrifuge tube. Avoid pipetting any
gel slices into pipet tips when collecting supernatant.
17. Repeat steps 13–15, combining the supernatant in the tube
from step 15.
18. Place the combined supernatant extract in the vacuum concentrator to remove acetonitrile and concentrate the peptide
digest (see Note 30).
19. Resuspend in MS loading solvent so that sample is roughly at a
concentration of 0.5–1 μg/μL (see Note 31).
20. Centrifuge samples at 14,000 Â g for 5 min.
21. Gently pipet the sample out of the tube, being sure not to
touch the bottom of the tube with the pipet tip (there will be a
small volume left in the tube), and transfer into an autosampler
vial. Make sure not to introduce any bubbles into the vial. Cap
and label vials.
22. Samples can be stored at À20
C until further processing for
LC-MS analysis.
3.6 In-Solution
Digestion Processing
for Bottom
up Proteomics (See
Note 24)
1. Precipitate 30 μg of protein sample with four volumes of prechilled acetone for 1 h at À20
C (see Note 32).
2. Pellet the protein by centrifugation at 15,000 Â g for 10 min at
4
C.
3. Carefully discard the supernatant and rinse the pellet with
300 μL of À20
C acetone.
4. Pellet the protein by centrifugation at 15,000 Â g for 10 min at
4
C.
5. Carefully discard the supernatant and let the pellet dry for
$5 min.
6. Dissolve the protein pellet in 15 μL of 8 M urea.
7. Sonicate the sample in the bath sonicator for 5 min.
8. Add 20 μL of 0.2% ProteaseMax surfactant solution.
9. Vortex for 5–10 min.
10. Add 58.5 μL of 50 mM ammonium bicarbonate.
542
Carolina Mehaffy et al.