Mapping of Phosphorylation Sites by LC-MS/MS - LC-MS/MS in Proteomics: Methods and Applications - page 130

12. Affinity gel wash: Wash the gel once with 300 Lofwater

to remove any residual wash/equilibration solution prior
to elution.
13. Sample elution: Place an end cap onto the column outlet
and place the column in a new collection tube.
14. Add 150

μ

L of 50 mM phosphate buffer A (pH 8.9) and
incubate for 3 min with shaking.
15. Elute by centrifugation and retain the flow-through.
Repeat the elution step once with 150

μ

μ L of 100 mM

phosphate buffer B (pH 8.9).
16. Purify phosphopeptides using Harvard (Ultra) microspin
columns according to manufacturer's instructions and ana-
lyze as soon as possible by LC-MS.

3.2. Isolation
ofPhosphopeptides
UsingTiO 2

Please note that the ratio of peptide to resin ( 7 ) aswellasthe
correct pH is critical ( see Note 3 ) in the binding step.
1. Place 1.25 mg of TiO 2 resin in a Mobicol spin column.
2. Wash the 1.25 mg TiO 2 resin with 200 μ L H2O.

3. Wash the 1.25 mg TiO 2 resin with 200 μ L methanol.

4. Equilibrate 1.25 mg TiO 2 resin with 300 μ L phthalic acid

solution.
5. Load 1-2 mg peptide dissolved in 300 L phthalic acid

solution onto the resin and incubate for 30 min with end-
over-end rotation.
6. Wash the resin twice with 300

μ

μ L phthalic acid solution.

7. Wash the resin twice with 300 μ L of 80% ACN and 0.1-

0.5% TFA solution.
8. Wash the resin twice with 300 μ L of 0.1-0.5% TFA

solution.
9. Elute phosphopeptides twice with 150

μ L of 0.3 M ammo-

nium hydroxide solution.
10. Adjust pH of eluent to 2.7 and clean phosphopeptides
using reverse-phase column (microspin columns; Nest
Group).
11. Dry sample under vacuum and analyze as soon as possible
by LC-MS.

3.3. Isolation
ofPhosphopeptides
UsingPAC:
Maleimide-
ContainingSolid
Phase
3.3.1.Synthesizing
Beads

1. Take 120 mol 3-maleimidopropionic

acid and dissolve in 0.8 mL dry DMF.

μ mol HoBT and 120 μ