Discontinuous pH gradient-mediated separation of TiO2-enriched phosphopeptides.

Global profiling of phosphoproteomes has proven to be a great challenge due to the relatively low stoichiometry of protein phosphorylation and poor ionization efficiency in mass spectrometers. Effective, physiologically relevant, phosphoproteome research relies on the efficient phosphopeptide enrichment from complex samples. Immobilized metal affinity chromatography and titanium dioxide chromatography can greatly assist selective phosphopeptide enrichment. However, the complexity of resultant enriched samples is often still high, suggesting that further separation of enriched phosphopeptides is required. We have developed a pH gradient elution technique for enhanced phosphopeptide identification in conjunction with titanium dioxide chromatography. Using this process, we demonstrated its superiority to the traditional "one-pot" strategies for differential protein identification. Our technique generated a highly specific separation of phosphopeptides by an applied pH gradient between 9.2 and 11.3. The most efficient elution range for high-resolution phosphopeptide separation was between pHs 9.2 and 9.4. High-resolution separation of multiply phosphorylated peptides was primarily achieved using elution ranges greater than pH 9.4. Investigation of phosphopeptide sequences identified in each pH fraction indicated that phosphopeptides with phosphorylated residues proximal to acidic residues, including glutamic acid, aspartic acid, and other phosphorylated residues, were preferentially eluted at higher pH values.