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:
- Buffer B1: 200mM NaCl, 35% MeCN, 0.4% TFA
- Buffer A2: 1M KH2PO4
- Buffer A1: 5% MeCN, 0.1% TFA
- Buffer B2: 50% MECN, 0.1% TFA
- 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.
- An additional 1.2 mL Buffer B1 is used to rinse the column to remove non-phosphorylated peptides.
- 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).
- The C18 column is then washed with 17.1 mL of Buffer A1.
- Peptides are eluted from the C18 column using 400 μL of Buffer B2, and this solution is lyophilized to dryness using a SpeedVac concentrator.