Identification of utilized phosphorylation sites in tagged proteins

Examination of utilized phosphorylation sites in an expressed, tagged protein requires a robust method that allows for maximum sequence coverage and identification of even low stoichiometry sites. The following protocol uses two to four enzymes to achieve high sequence coverage. Each of these digestions is analyzed un-enriched (C18) and enriched by TiO chromatography. By using this strategy and the high dynamic range and mass accuracy of the LTQFT hybrid linear ion trap – FTICR, sites modified at least down to the 0.1% level can be determined.