Biomedical Engineering Reference
In-Depth Information
6. Wash beads sequentially with 200
μ
lTiO
2
loading buffer,
then with 200
lTiO
2
washing solution A (80% ACN/5%
TFA) and finally with 200
μ
μ
lTiO
2
washing solution B (10%
ACN). Discard the resulting supernatant in each case.
7. Elution of bound phosphopeptides is achieved by incubat-
ing the beads with 50
lTiO
2
elution solution A (ammonia
water, pH 11) (
see
Section
2.4
) for 5 min, spinning and col-
lecting the supernatants (
see
Note 27
).
8. Beads are subjected to a second elution round with TiO
2
elution solution B (
see
Note 28
). Combine this elution
with the previous one to obtain the final phosphopeptide-
enriched sample.
9. Acidify eluates with 10% FA final concentration, lyophilise
and resuspend in 10
μ
l 0.1% TFA for analysis by
LC-MS/MS (
see
Note 29
).
μ
1. The identification of phosphopeptides by mass spectrometry
can be performed using, for example, a quadrupole time-of-
flight (Q-TOF) mass spectrometer connected on line to a
nanoflow ultrahigh-pressure liquid chromatograph.
2. HPLC separations can be done using a BEH reverse-phase
column. The suggested mobile phases are solution A (0.1%
FA in LC-MS grade water) and solution B (0.1% FA in
LC-MS grade ACN).
3. The recommended gradient run is from 1% B to 35% B in
100 min followed by a 5-min wash at 85% B and a 7-min
equilibration step at 1% B (
see
Note 30
).
4. Perform a mass spectrometry data-dependent analysis
(DDA) in which the three most abundant multiply charged
ions present in the survey spectrum are automatically mass
selected and fragmented by collision-induced dissociation in
each cycle. MS scans of 500 ms, followed by three MS/MS
scans of 1 s each are suggested to be acquired within a mass
range of 50-2,000
m
/
z
(
see
Note 31
).
5. The phosphopeptide sequences can be identified by loading
the MS/MS data files in a protein search engine, such as
Mascot, Seaquest and Phoenix, among others.
3.5. Identification
ofPhosphopeptides
byLC-MS
4. Notes
1. This assay can be performed with any cell line to be stud-
ied. Here we exemplify the approach by using the murine
NIH-3T3 fibroblasts as a cell model commonly used in
cell-signalling studies.
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