Mapping of Phosphorylation Sites by LC-MS/MS - LC-MS/MS in Proteomics: Methods and Applications - page 128

Please note the subdivision between the different packing materi-
als, glass beads, and dendrimers.
1. Bio-Spin column (Bio-Rad).
2. Anhydrous N , N -dimethylformamide, 99.8% (Sigma-

Aldrich).
3. Aminopropyl CPG beads (Proligo).
4. N -Hydroxybenzotriazole (HoBT) (Nova Biochem).
5. 3-Maleimidopropionic acid, 97% (Sigma-Aldrich).
6. N , N -Diisopropylcarbodiimide, 99% (Sigma-Aldrich).
7. Anhydrous dichloromethane, 99.8% (Sigma-Aldrich).

8. Ninhydrin reagent solution (Sigma-Aldrich).
9. Acetyl chloride,

≥

≥

99% (Sigma-Aldrich).
10. Anhydrous methanol, 99.8% (Sigma-Aldrich).
11. Imidazole (Sigma-Aldrich).
12. Anhydrous acetonitrile, 99.8% (Sigma-Aldrich).
13. N -(3-Dimethylaminopropyl)- N

-ethylcarbodiimide
hydrochloride (EDC) (Sigma-Aldrich).
14. Cystamine (Sigma-Aldrich).
15. MES, low moisture content, ≥ 99% (titration) (Sigma-

Aldrich).
16. Empty spin columns (MoBiTec).
17. Filter for spin columns (MoBiTec).
18. Glass tubes (Waters).
19. Trifluoroacetic acid (TFA) (Pierce).
20. Tris(2-carboxyethyl) phosphine hydrochloride (TCEP)
(Pierce).
21. For 1 mg of peptide: Sep-Pak R

Vac C18 cartridge

(Waters).
22. For micrograms of peptide: MicroSpin TM Columns (Nest
Group).

3. Methods

3.1. Isolation
ofPhosphopeptides
UsingIMAC

The isolation of phosphopeptides using IMAC can be done on
peptides with and without methyl esterification. Methyl ester-
ification reduces the amount of non-specific binding of non-
phosphopeptides. However, as a first step, choose the phospho-
peptide enrichment without the methyl esterification. Please note
that the ratio of peptide to resin ( 7 ) aswellasthecorrectpHis
critical ( see Note 3 ) in the binding step.