Biology Reference
In-Depth Information
4. Then 60
L trypsin sequencing grade are added.
If the succinylated peptide library beads are used the amount
of trypsin should be four times larger.
5. The bead suspension is vortexed and incubated overnight at
37 °C under gentle shaking.
6. 200
μ
L of 0.2
μ
g/
μ
L of 500 mM formic acid are then added; the suspension
is vortexed, for few seconds, and incubated for about 40 min
at room temperature.
7. Then the supernatant is recovered by fi ltration (30,000
MWCO) under centrifugation (microfuge, at least 5,000
μ
g
for 20 min) in order to separate insoluble material and beads.
8. In order to fully extract the remaining peptides the beads are
washed under centrifugation once with 50
×
μ
L of 500 mM for-
mic acid and mixed to previous fi ltrate.
9. Stripped beads could then be kept at −20 °C for possible fur-
ther analysis.
10. The solution of peptides is then dried by speedvac and redis-
solved in 20
μ
L HPLC solvent for LC-mass spectrometry
analysis.
As a possible alternative to this protocol,
1. 100
L of a solution composed of 8 M
urea, 100 mM Tris (hydroxyethylamine) pH 8.5, and 5 mM
tris (2-carboxyethyl)phosphine. After few seconds of vortex-
stirring the suspension is agitated gently for 30 min at room
temperature.
2. Cysteine residues are then acetylated by addition of 10 mM
iodoacetamide and incubated in the dark for at least 15 min.
3. The bead suspension is then diluted with 100 mM Tris-
(hydroxyethylamine) pH 8.5 to reach a fi nal concentration of
2 M urea.
4. Trypsin is added (20
μ
L are added with 600
μ
L, this corresponding
roughly to 1:50 protease/protein ratio) along with CaCl
2
to
1 mM concentration and the resulting mixture incubated over-
night at 37 °C.
5. The obtained peptides are separated from the beads by centrifuga-
tion at 1,000 ×
g
and stored at −80 °C until the day of analysis.
μ
g as 0.5
μ
g/
μ
6. Before analysis the peptide solution is acidifi ed to 5 % formic acid
and centrifuged at 18,000 ×
g
to remove possible solid material.
Proteomic analysis may not be compatible with the protein solution
after classical treatment with CPLL. In this case two possibilities are
available: (1) the collected samples are put in appropriate buffers or
(2) elutions are customized to comply with the following analysis.
In the fi rst case the protein samples are desalted by extensive dialysis
3.6 Interrelation
Between Elution
and Analysis
