Optimized Protocol for On-Target Phosphopeptide Enrichment Prior to Matrix-Assisted Laser Desorption-Ionization Mass Spectrometry Using Mesoporous Titanium Dioxide

A novel on-target phosphopeptide enrichment method is presented that allows specific enrichment and direct analysis by matrix assisted laser desorption-ionization mass spectrometry (MALDI-MS) of phosphorylated peptides. Spots consisting of a thin film of anatase titanium dioxide are sintered onto a conductive glass surface. Enrichment and analysis can be performed on the modified target with minimal sample handling. The protocol leads to an enrichment efficiency that is superior to what has been reported before for similar methods. The method was tested using beta-casein as a model phosphorylated protein as well as with a custom peptide mixed with its phosphotylated form. A very low detection limit, a significantly improved phosphoprofiling capability, and a simple experimental approach provide a powerful tool for the enrichment, detection, and analysis of phosphopeptides.