Biology Reference
In-Depth Information
3. Sample digestion: Dilute trypsin to a concentration of 25 ng/
μ
L per gel piece
(depending on intensity) and keep on ice for 1 h. Add 20-30
L in 50 mM NH
4
HCO
3
and add 3-10
μ
L
50 mM NH
4
HCO
3
(depending on size of the gel piece) and
incubate at 37 °C overnight.
4. Peptide extraction: Sonicate the gel pieces in the surrounding
solution and collect the liquid. If the volume has dramatically
decreased, add few more
μ
L of 50 mM NH
4
HCO
3
before son-
ication. Dry the liquid down in a SpeedVac and store the sam-
ples at −20 °C until analysis.
μ
The TiO
2
material is provided by the manufacturer packed in tips,
Spin Tip (or can be produced in house [
31
]) and should be han-
dled using a centrifuge.
See
Note 7
for specifi c enrichment of tyro-
sine phosphorylated peptides.
3.3.5 Enrichment
of Phosphopeptides
1. Put the Spin Tip in a punched lid of an Eppendorf tube that
will be used as waste tube. Check that the tip does not meet
the bottom of the tube.
2. Add 20
L of loading buffer A and then centrifuge at 3,000 ×
g
,
room temperature, for 2 min to condition the TiO
2
material.
3. Add 20
μ
L of loading buffer B and centrifuge 3,000 ×
g
, room
temperature, for 2 min to equilibrate the TiO
2
material.
Remove the 40
μ
L collected in the waste tube.
4. To adsorb the peptides, dissolve desalted peptides in 100
μ
L
loading buffer B and add this to the Spin Tip. Centrifuge at
1,000 ×
g
, room temperature, for 10 min. Collect the solution
that passes through the Spin Tip and put it back into the Spin
Tip again. Centrifuge at 1,000 ×
g
, room temperature, for
10 min. Remove the liquid collected in the waste tube.
5. Rinse the peptides by adding 20
μ
L of loading buffer B and
centrifuge at 3,000 ×
g
, room temperature, for 2 min. Rinse by
again by adding 20
μ
L of loading buffer A and an additional
centrifugation at 3,000 ×
g
, room temperature, for 2 min.
6. Discard the waste tube and put the Spin Tip into a new
Eppendorf tube. Elute the adsorbed peptides by adding 50
μ
L
of elution buffer A and centrifuge at 1,000 ×
g
, room tempera-
ture, for 5 min. Then add 50
μ
L of elution buffer B and cen-
trifuge at 1,000 ×
g
, room temperature, for 5 min.
7. Acidify the peptides by adding 100
μ
L of 20 % TFA. Confi rm
the acidic pH by pH-paper. Then, desalt the peptides accord-
ing to the StageTip procedure described in Subheading
3.3.1
.
To check sample quality,
see
Note 4
.
μ
Here we present the protocol for using a 7-tesla LTQ-FT Ultra
tandem mass spectrometer modifi ed with a nano electrospray ion
3.4 LC-MS Analysis
