Enrichment of Phosphorylated Peptides Using Titanium Dioxide

The starting material for the phosphopeptide enrichment is generally a tryptic in solution digest of a complex mixture. At our resource, Titanium dioxide enrichment is conducted on an ÄKTA Purifier. Peptides are enriched using 5 μm titanium dioxide beads (GL Sciences, Tokyo, Japan) packed into an analytical guard column with a 62 μL packing volume (Upchurch Scientific, Oak Harbor, WA USA). The following buffers are used:

1. Buffer B1: 200mM NaCl, 35% MeCN, 0.4% TFA
2. Buffer A2: 1M KH2PO4
3. Buffer A1: 5% MeCN, 0.1% TFA
4. Buffer B2: 50% MECN, 0.1% TFA

TiO2 Chromatography

1. Typically 2mg of dried peptide pellets are resuspended in 240 μL of Buffer B1 and injected via an autosampler over the titanium dioxide column at a flow rate of 100 µL/ min.
2. An additional 1.2 mL Buffer B1 is used to rinse the column to remove non-phosphorylated peptides.
3. Phosphorylated peptides are eluted from the titanium dioxide column using 15 mL of Buffer A2 (1M KH2PO4). A switching valve is used to direct these elutions onto a C18 macrotrap column (Michrom Bioresources, Auburn, CA, USA).
4. The C18 column is then washed with 17.1 mL of Buffer A1.
5. Peptides are eluted from the C18 column using 400 μL of Buffer B2, and this solution is lyophilized to dryness using a SpeedVac concentrator.