Titanium Dioxide Enrichment Protocol

Loading Buffer:

• 0.5%-2% TFA in 50%-80% ACN
• I used 1% TFA in 80% ACN

Wash Buffer:

• The same as loading buffer, but make sure to keep in a separate eppie.

Intermediate Buffer:

• 0.1% TFA in 80% ACN.
• This step brings the pH back to neutral

Elution Buffer:

• 500 mM NH4OH in H2O

Steps:

1. Adequate 6ul of elution buffer to a new eppie.
2. Condition TiO2 tip in loading buffer by pipetting 10 times slowly.
3. Load sample by pipetting several times.
4. Wash the tip in the wash buffer by pipetting several times.
5. Bring the pH back to neutral by pipetting into the intermediate buffer 3-5 times.
6. Elute the peptides into the pre-alequated elutant eppie.
7. Speed-vac to dry the sample.
8. Reconstitute in 5% Formic Acid.
9. Mass Spec it!