Biology Reference
In-Depth Information
5. Re-extract by adding 500
l of new extraction buffer.
6. Centrifuge for 3 min, 6,000 ×
g
at 4 °C.
7. Transfer the phenolic phase into a new 2 ml tube and precipi-
tate the proteins overnight (or at least 150 min) with 5 vol-
umes 100 mM ammonium acetate in methanol at -20 °C.
8. Centrifuge 60 min, 16,000 ×
g
, at 4 °C.
9. Remove the supernatant and rinse the pellet twice (do not
resuspend) in 2 ml rinsing solution (cold acetone/0.2 % DTT).
After the fi rst rinse leave in rinsing solution for 1 h at −20 °C.
After rinsing, centrifuge 30 min, 16,000 ×
g
, at 4 °C.
10. Dry the pellet at RT.
11. Suspend the pellet in 100
μ
μ
l lysis solution (optimum concen-
l).
12. Clear samples by centrifugation (16,000 ×
g
, 2 ×
g
30 min at
18 °C).
13. Quantify the samples (
see
Note 4
).
tration is 1-5
μ
g/
μ
1. From the quantifi cation analysis calculate the amount of
μ
l
5.2 Protein Digestion
and Cleanup
necessary to digest 50-60
μ
g protein.
2. Add
x
l 0.5 M IAA to reach a fi nal concentration of 0.015 M
and incubate for 0.5 h in the dark at RT (shaker) (
see
Note 5
).
3. Add
x
μ
l 100 mM ABC to reach a fi nal concentration of 75 mM
(
see
Note 5
).
4. Add 3
μ
μ
l trypsin (0.2
μ
g/
μ
l) and incubate at 37 °C (overnight).
5. Add
x
l of 10 % TFA to reach a fi nal concentration of 0.5 %
(
see
Notes 5
and
6
).
6. Wash SPE columns with 1 ml 95 % ACN.
7. Equilibrate SPE columns with 1 ml 2 % ACN 0.1 % TFA.
μ
8. Apply the sample to the column.
9. Wash SPE columns with 1 ml 2 % ACN 0.1 % TFA.
10. Elute the sample with 84 % ACN 0.1 % TFA.
11. Dry the sample in the speed vac.
12. Dissolve in 40
μ
l 0.1 M AF.
13. Vortex 60 min.
14. Centrifuge 5 min 16,000 ×
g
.
15. Determine the peptide concentration through measurement at
A280 nm (NanoDrop, Thermo Scientifi c, Waltham, MA, USA)
(
see
Note 4
).
5.3 Peptide
Separation, MS
and Identifi cation
1. Inject 2.5
g of digested proteins on the fi rst RP column.
2. Elute the sample from this column stepwise under high pH
and ultrahigh pressure at 2
μ
l/min. Samples are subsequently
eluted in fractions of 12, 15, 18, 20, 25, 35, and 65 % ACN in
μ
