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. 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). Dried peptide pellets are resuspended in 250 μL wash solution (35% acetonitrile, 4.5% TFA) and injected via an autosampler over the titanium dioxide column at a flow rate of 50 µL/ min with an additional 3.9 mL wash solution to remove non-phosphorylated peptides. This is then followed by 3.5 mL of rinse solution (0.1% TFA). Phosphorylated peptides are eluted from the titanium dioxide column directly onto a C18 macrotrap peptide column (Michrom Bioresources, Auburn, CA, USA) using 15 mL of elution solution (1M KH2PO4). The C18 column is then washed with 17.1 mL of rinse solution. Peptides are eluted from the C18 column using 400 μL of organic elution solution (50% acetonitrile, 0.1% TFA), and this solution is lyophilized to dryness using a SpeedVac concentrator.