Identification of sites of phosphorylation
LC-MS/MS analysis of the TiO2 enriched phosphopeptides, as well as the flow through fraction containing all other peptides, is done on a LTQ Orbitrap XL instrument using multistage activation. Database searching is performed using the Mascot search algorithm (http://www.matrixscience.com/home.html).
Figure 1 demonstrates how the TiO2 enrichment reduced the complexity of this sample thereby enhancing the observation of the indicated phosphopeptides.
Requirements: >30ng proteins of interest purified in a 1 or 2D gel band or immunoprecipitated in solution with minimal interference of antibody proteins