46 A Practical Guide to Cancer Systems Biology
- Hydroxy-acid modified metal oxide chtomatography
(HAMMOC)^3
Materials:
- Lactic acid (LA) (CAS NO. 50-21-5)
- Piperidine (CAS NO. 110-89-4)
- Trifluoroacetic acid (TFA) (CAS NO. 76-05-1)
- Phosphoric acid (CAS NO. 7664-38-2)
- Acetonitrile (ACN), HPLC grade
- 1.5 mL microcentrifuge tube (#0030 120.086, Eppendorf)
- Pipette tips
- C8 StageTips in 1.5 mL microcentrifuge tube
- SDB-XC StageTips in 1.5 mL microcentrifuge tube
- Methanol (MeOH)
Procedure:
I. Buffer preparation:
Solution A: 0.1% TFA, 80% ACN
Solution B: 0.1% TFA, 5% ACN
Solution C: 300 mg/mL LA
Solution D: 0.5% piperidine
TiO 2 packing
II. Take TiO 2 from the desiccator and weight the TiO 2 spheres in a 1.5 mL
tube (0.5 mg TiO 2 /tip).Hint:TheweightedTiO 2 should be higher than the required amount.
For example, if you need 2 mg TiO 2 , prepare 2.5–3 mg TiO 2.III. Add MeOH to the TiO 2 tube (40μLMeOH/mgTiO 2 .Ex.for1.5mg
TiO 2 ,add60μL MeOH). Put a tiny stir bar into the tube. Wash the
stir bar with MeOH before use.
IV. Allow TiO 2 and MeOH to be continuously mixed on a plate stirrer.
Transfer 20μlTiO 2 (0.5 mg) solution to each C8 StageTip.
V. Centrifuge at 500×g for 3–5 minutes to remove MeOH.
Phosphopeptide enrichment
VI. Preconditionthe TiO 2 tips by loading 20μLsolutionB, centrifuge
at 1,500×gfor2minutes.