Biology Reference
In-Depth Information
the protein from a preparative gel (i.e., a gel containing more
than 90
g of proteins to obtain enough material for MS spec-
tra acquisition). Depending on the excision mode (automated
or manual), the use of Bind-Silane-coated glass plate has to be
considered. A gel containing more or less 300
μ
g of proteins is
run; the IEF step is performed on the same pH range, but with
an increased amount of total volt per hour (e.g., 90,000 V h for
24 cm on a pH 4-7). The SDS-PAGE is carried out in the same
conditions. This preparative gel is then stained with Coomassie
blue or a fl uorescent post-migration stain (
see
Note 12
).
μ
4. After the scanning of the preparative gel and matching with
analytical ones, a pick list is generated and used with the auto-
mated picker (e.g., here the Ettan Picker; GE Healthcare).
Excised gel plugs are collected in microplates and used for pro-
tein identifi cation protocols (
see
Note 13
).
5. In this example, the protocol described is automatically per-
formed on the Ettan Spot Handling Workstation (GE
Healthcare), composed of an Ettan Picker, a digester, and a
spotter, in a closed cabinet to avoid contamination with kera-
tin, for example (
see
Note 14
). The digestion is performed in
a temperature-controlled closed microplate tower. Washing
steps aiming at removing interfering substances on the gel
plugs such as Coomassie blue and excess of SDS (providing
high background on the MS spectra) are then performed with
the washing solution (150
L, two or three times for 20 min
depending on the intensity of the staining if Coomassie blue
has been used). A solution of 75 % acetonitrile is then added in
the microplate wells for 30 min, then removed, and the gel
plugs are left to dry at room temperature.
6. Protein contained in the gel plug is digested by the addition of
trypsin or another proteinase; here 6
μ
L of trypsin solution is
added to the dried gel piece, the microplate is covered, and the
digestion is performed at 37 °C for 4 h.
7. Extraction of peptides: 35
μ
L of peptide extraction solution is
added in the well at the end of the digestion step and the
supernatant is recuperated in a new microplate. This step is
repeated twice and the supernatant pooled with the previous
one. Peptides are then concentrated by drying.
8. Peptides are resuspended in 2
μ
L sample-spotting solution and
mixed in the microplate wells. Then, 0.7
μ
μ
L of this mixture is
L of matrix solution is added
on top (with three mixing strokes in the spotter needle before
fi nal deposition).
9. Once the peptides are on the MALDI target, the instrument is
calibrated with a mixture of known peptides and the acquisi-
tion of spectra (MS and MS/MS) is started.
put on a MALDI target, and 0.7
μ
