Improved Enrichment Strategies for Phosphorylated Peptides on Titanium Dioxide Using Methyl Esterification and pH Gradient Elution

摘要: Improvements to phosphopeptide enrichment protocols employing titanium dioxide (TiO2) are described and applied identification of phosphorylation sites on recombinant human cyclin-dependent kinase 2 (CDK2). Titanium binds phosphopeptides under acidic conditions, they can be eluted basic conditions. However, some nonphosphorylated peptides, particularly bind elute these conditions as well. These peptides contribute significantly ion suppression also increase sample complexity. We show here that the conversion peptide carboxylates their corresponding methyl esters sharply reduces nonspecific binding, improving selectivity for phosphopeptides, just has been reported immobilized metal affinity chromatography (IMAC) columns. present evidence monophosphorylated effectively fractionated from multiply phosphorylated well via stepwise elution TiO2 using pH step gradients 8.5 11.5. approaches were CDK2 in vitro by yeast CAK1p absence cyclin. confirmed at T160, a site previously documented shown necessary activity. we discovered several novel partial S46, T47, T165, Y168 when ion-suppressing eliminated new protocols.