Mapping of Phosphorylation Sites by LC-MS/MS - LC-MS/MS in Proteomics: Methods and Applications - page 132

15. Wash once with 0.1% TFA.
16. Add 2 mL of 10 mM TCEP in 100 mM phosphate buffer
(pH 6.0); the pH is crucial!
17. Press halfway through, incubate for 8 min at room temper-
ature.
18. Wash with 0.1% TFA.
19. Elute with 750

μ L of 80% ACN, 0.1% TFA.

20. Add 100 L phosphate buffer, check and adjust pH to 6.3

and partly remove ACN to a final of 30% in a speedvac
device.
21. Take 5-10 mg of prepared maleimide beads, place in
blocked Mobicol column.
22. Add solution with the derivatized peptides (pH 6.3) to the
beads.
23. Incubate for 1 h at room temperature.
24. Wash the beads under shaking two to three times with 280
μ

μ

L each of 3 M NaCl, water, methanol, and 80% ACN.
25. Resuspend the beads in 80% ACN and transfer to glass
tube.
26. Remove excess ACN (supernatant).
27. Incubate for 1 h in 10% TFA and 30% ACN (phosphopep-
tide recovery).
28. Spin tube down and transfer supernatant to new glass tube.
29. Add 40% ACN to beads, vortex, spin down, and take out
the supernatant.
30. Add 40% ACN to beads, vortex, spin down, and take out
the supernatant.
31. Pool all supernatants.
32. Dry under vacuum.
33. Resuspend in 0.1% TFA and 0.005% phosphoric acid for
MS analysis.

This section describes the isolation of phosphopeptides also using
phosphoramidite chemistry. Instead of derivatized glass beads, a
dendrimer is used to capture the phosphopeptides. Methylation of
peptides can be performed as described in Section 3.1.1 . Before
use, adjust pH of dendrimer to 6. Dry under vacuum and re-
dissolve in reaction solution.
1. The methylated peptide sample is dissolved in a solution of
40

3.3.3. Isolation
ofPhosphopeptides
UsingPAC:Dendrimer

μ L of methanol, 40 μ L of water, and 80 μ L of acetoni-

trile.
2. Dissolve peptide methyl esters in 250 L of a reaction solu-

tion containing 50 mM EDC, 50 mM imidazole (pH 6.5),

μ