- Phosphoproteome: Sample Preparation 43
XII. Centrifuge at 15,700 g for 2 minutes at room temperature and remove
the supernatant carefully.Hint: Do not remove the white disc between the organic and
aqueous layers. It is acceptable to remove a few microliters of
samples.XIII. Vacuum dry the peptide samples.
XIV. Dissolve the peptides by adding 300− 500 μLsolutionA. The precipi-
tation from white disc cannot be dissolved.
XV. Peptide desalting using EmporeTMSDB-XC 6 mL cartridge according
to the manufacturer’s instructions.
Hint: Load all the samples including precipitations to the desalting
column to increase the recovery of peptides.- Dimethyl labeling of peptides^12
Materials:
- 1M TEAB
- 37% formaldehyde solution (CH 2 O, CAS NO. 50-00-0)
- 20% formaldehyde-13C, D2 solution (D^13 CDO, CAS NO. 63101-50-8)
- Cyanoborohydride (NaBH 3 CN)
Hint:StoreNaBH 3 CN in a desiccator.- 25% ammonia
- Formic acid
Procedure:
I. Buffer preparation (freshly prepared):
i. 100 mM TEAB
ii. 4% CH 2 O
iii. 4%^13 CD 2 O
iv. 0.6 M NaBH 3 CNII. Dissolve 100μg peptides by 100μL 100 mM TEAB (100μLcanbeused
for a maximum of 100μgpeptides).