Biology Reference
In-Depth Information
10. Trypsin solution was prepared in the buffer provided (Promega)
to 1
L
50 mM NH
4
HCO
3
. This should be enough for 20 digestions
at a concentration of 12.5 ng/
μ
g/
μ
L, and then 5
μ
L of the solution was added to 395
μ
L. An adequate volume of
trypsin solution was added to cover the gel bands. This volume
will vary but is usually around 20
μ
μ
L and up to 50
μ
L for large
gel bands.
11. The gel pieces were rehydrated at 4 °C for at least 30 min in
trypsin solution. Incubation must be kept cool so as to allow as
much active trypsin to be absorbed into the gel as possible
before auto-digestion occurs (
see
Note 7
).
12. The tubes were centrifuged briefl y, and an additional 25
μ
L
50 mM NH
4
HCO
3
was added to cover gel pieces.
13. Proteins were digested overnight at 37 °C, or for a minimum
of 4 h.
14. The digest solution supernatant (if any) was transferred into a
clean 0.65 mL tube.
15. Peptides were extracted from the gel pieces with 30
L (or
enough to cover) of 50 % ACN (v/v) and 2 % (v/v) formic acid
and incubated for 20 min with vortexing at room temperature.
The tubes were centrifuged briefl y, and the supernatant was
removed and combined with initial digest solution supernatant.
For large pieces of gel, use more liquid where required.
μ
16.
Step 15
was repeated to give a combined peptide extract vol-
ume of around 60-100
L (
see
Note 8
). For large gel slices,
use slightly larger volumes, repeat extraction a third time, and
combine all extracts.
17. The extracted digests were vortexed, centrifuged briefl y, and
then concentrated in a vacuum centrifuge to approximately
5
μ
L each. Do not dry completely if possible. The peptides
were reconstituted to a volume of 10
μ
L with 1 % (v/v) formic
acid. If peptides are reduced to dryness, reconstitute peptides
in 10
μ
L of 1 % (v/v) formic acid (
see
Note 9
).
18. The 10
μ
L extracts were centrifuged at 17,000 ×
g
for at least
15 min to pellet any microparticulates. The supernatant was
very carefully transferred to a fresh 0.65 mL polypropylene
tube, or into a 96-well plate. The sample is now ready for anal-
ysis by LC-MS/MS.
μ
3.3 nanoLC-MS/MS
of Gel Fractions
1. Each of the 16 fractions prepared was analyzed sequentially
using a nanoLC-MS/MS system, employing an LTQ-XL ion-
trap mass spectrometer (Thermo, San Jose, CA).
2. Reversed phase columns were packed in-house to approxi-
mately 7 cm (100
m i.d.) using 100 Å, 5 mM Zorbax C18
resin (Agilent Technologies, CA, USA) in a fused silica capil-
lary with an integrated electrospray tip (
see
Note 10
).
μ
