In-Depth Analysis of Protein Phosphorylation by Multidimensional Ion Exchange Chromatography and Mass Spectrometry - LC-MS/MS in Proteomics: Methods and Applications - page 119

2. Centrifuge the samples at 1,000 g for 5 min. Take aside

the supernatant (unbound fraction) for further enrichment
with TiO 2 beads.
3. Beads are now washed with 300

×

lofIMACload-
ing/washing buffer (50% ACN+ 0.1% TFA).
4. Combine the unbound and washed fraction as the I1 frac-
tion for later will TiO 2 enrichment ( see Note 22 ).
5. For the elution of non-phosphorylated acidic peptides
and less acidic phosphopeptides (mainly monophospho-
peptides), treat beads with 300

μ

l IMAC elution solution
A (20% ACN, 1% TFA) for 5 min. Spin down and keep
the supernatant as the I2 fraction. This will also be further
enriched with TiO 2 beads ( see Note 23 ).
6. Highly acidic phosphopeptides, enriched in multiphospho-
peptides (I3 fraction), are obtained by eluting with 300

μ

l
IMAC elution solution B (ammonia water, pH 11.3, pre-
pared as indicated in Section 2.4 ) for 5 min. The super-
natant obtained after centrifugation constitutes the I3 frac-
tion. Repeat this step twice and combine the two ammonia
water elutions.
7. Wash the beads with 50

μ

l of IMAC elution buffer C
(ammonia water, pH 11.3, containing 50% ACN). Com-
bine the resulting supernatant with the previous ammonia
elutions rendering the final I3 fraction ( see Note 24 ). At
this stage the beads can be discarded.
8. Ammonia fraction (I3) should be acidified with 10% FA
final concentration. In our example, we will use 65

μ

μ l 100%

FA for I3 acidification.
9. Lyophilise I2 and I3 fraction.
10. Resuspend the I3 fraction in 10 μ l of 0.1% TFA for analysis

by LC-MS.

1. Resuspend the TiO 2 beads with 100% ACN to 50% slurry.
Use 10

3.4.2.TitaniumDioxide
(TiO 2 )Chromatography

l of beads per sample ( see Note 25 ).
2. Spin the beads down and remove the ACN supernatant.
3. Wash the beads first with 200

μ

μ l of water and then twice

lTiO 2 loading buffer (1 M glycolic acid in 80%
ACN, 5% TFA) ( see Note 26 ).
4. Resuspend I2 fraction with 2

with 200 μ

μ l4Murea,3 μ l1%SDSand

lTiO 2 loading buffer.
5. Incubate factions I1 and I2 with 10

200 μ

lTiO 2 beads
for 30 min at room temperature. After this time, cen-
trifuge the samples at 1,000

μ

×

g for 5 min and discard the

supernatant.