Reagents and materials

1. 80% ACN with 6% TFA (!!make fresh everyday!!)
2. 80% ACN
3. 80% ACN with 0.5M glycolic acid (or lactic acid)
4. 50% ACN with 1% ammonium hydroxide (make fresh)
5. Ti-IMAC beads (MR-TIM005from ReSyn Biosciences)
6. magnet

Note: Use 50 to 100 µL of Ti_IMAC beads for every 1 mg of peptide. 50 µL will be cleaner, but 100 µL will have higher phosphopeptide yield.

Procedure

1. Wash beads three times with 1 mL 80% ACN with 6% TFA
2. Resuspend sample in 1 mL 80% ACN with 6% TFA and combine with beads
3. Vortex 20 minutes at ambient temperature
4. Wash beads three times with 1 mL 80% ACN with 6% TFA
5. Wash beads once with 1 mL 80% ACN
6. Wash with 1 mL 80% ACN with 0.5 M glycolic acid
7. Wash beads one times with 1 mL 80% ACN
8. Elute two times with ~300 µL 50% ACN with 1% ammonium hydroxide
9. Dry and desalt