Mapping of Phosphorylation Sites by LC-MS/MS - LC-MS/MS in Proteomics: Methods and Applications - page 131

2. Add 120 μ mol diisopropylcarbodiimide to make the link

solution.
3. Place 100 mg amino glass beads (5 mg good for 1 phospho-
peptide isolation) into washing columns.
4. Wash with two volumes of DMF.
5. Add the link solution to the beads, close the column, and
incubate for 90 min at RT with end-over-end rotation.
6. Wash beads three times with DMF and then two times with
CH 4 Cl 2 .
7. Resuspend in CH 4 Cl 2 , transfer to tube, and remove the
supernatant.
8. Dry under vacuum.

The following is for 1-3 mg peptide starting material. For smaller
starting amounts, the columns and volumes must be adjusted
accordingly.
1. Add 160

3.3.2.Phosphopeptide
Isolation

L dry acetyl chloride to 1 mL dry anhydrous
methanol (methanolic HCl).
2. Reconstitute 1 mg dry peptides in 500

μ

L-1 mL methano-
lic HCl (for less peptide use, corresponding amount of
methanolic HCl).
3. Incubate for 120 min at 12 ◦ C under dry conditions
(methyl esterification).
4. Dry the sample quickly under vacuum in a cold Speedvac.
5. Dissolve the sample in 40

μ

μ L methanol, 40 μ Lwater,and

L ACN prior to derivatization.
6. Place 5 mg EDC (final concentration of 50 mM) and
225 mg cystamine (final concentration of 2 M) into an
Eppendorf tube.
7. Add imidazole to 50 mM and MES to 100 mM.
8. Adjust pH to 5.8 with micro pH meter and fill up with
water to 500

80 μ

μ L.

L reaction solution to the peptides, verify that
pH is between 5.5 and 5.8.
10. Shake vigorously for 8 h at room temperature. Do not
exceed this time as otherwise the formation of side prod-
ucts will significantly increase.
11. Wash Sep-Packs columns (use appropriate column) with
one volume of methanol, then 80% ACN, then 0.1% TFA.
12. Acidify derivatized phosphopeptides to pH 2.7.
13. Load derivatized peptides onto spin column.
14. Load the flow-through once more.

9. Add 250 μ