Protocol for Quantitative Proteomics of Cellular Membranes and Membrane Rafts - LC-MS/MS in Proteomics: Methods and Applications - page 244

5. Rehydrate the gel pieces with 100 L of the 5 mM TCEP

working solution and incubate on a block heater for 45 min
at 55 ◦ C to reduce the proteins.
6. Remove the 5 mM TCEP solution using a pipette and
dehydrate the gel pieces to small white clumps by washing
twice with acetonitrile, removing the solutions after washes
using a pipette, then dry the gel pieces fully in a vacuum
centrifuge for 5 min.
8. Prepare a 0.1 M MMTS stock solution immediately before
use by diluting 10.3

μ

Liso-
propanol in a glass vial ( see Note 6 ). MMTS is a relatively
dense liquid and will collect at the bottom of the vial. Mix
the solution thoroughly by careful aspiration with a pipette.
Prepare a 10 mM working solution of MMTS by diluting
150

μ L of MMTS with 989.7 μ

L of the 0.1 M MMTS stock solution with 1.35 mL
50 mM TEAB.
9. Rehydrate the gel pieces with 100

μ

L of the 10 mM
MMTS working solution and incubate at room tempera-
ture for 30 min.
10. Remove the 10 mM MMTS working solution using a
pipette.
11. If the gel pieces are not completely destained by this stage,
incubate the gel pieces in 50% acetonitrile/50 mM TEAB
on a heating block at 37 ◦ C with shaking until destained
then remove the solution using a pipette.
12. Dehydrate the gel pieces to small white clumps by wash-
ing with acetonitrile, remove the wash solutions using a
pipette, dry the gel pieces fully in a vacuum centrifuge for
5 min, and then place on ice.
13. Prepare a trypsin stock solution by dissolving 25

μ

μ g

lyophilised trypsin in a 250 L solution of 0.1% TFA in

water. This solution can be aliquoted and stored at -20 ◦ C
for up to 6 months. Immediately before use, prepare a
trypsin working solution by diluting a 30

μ

μ L aliquot of

the trypsin stock solution with 200 μ L TEAB chilled on

ice ( see Note 17 ).
14. Add just enough trypsin working solution to the gel pieces
to cover the estimated volume of the fully hydrated gel
pieces. Incubate the suspension on ice for 30 min to rehy-
drate the gel pieces with trypsin solution. After rehydra-
tion, remove any excess trypsin solution using a pipette.
Cover the gel pieces with 50 mM TEAB and incubate at
37 ◦ Cfor4h( see Note 18 ).
15. Transfer the digest solution from the gel piece suspension
to a clean centrifuge tube.