Mesoporous TiO2 Nanocrystal Clusters for Selective Enrichment of Phosphopeptides

Protein phosphorylation plays a key role in most cellular processes. Studying phosphopeptides in complex biological samples has been a great challenge due to their low abundance as well as the coexistence of excessive amounts of salts or surfactants. In this work we demonstrate a general approach for selective separation of phosphopeptides using a class of novel mesoporous nanostructured materials. TiO2 nanocrystals are first self-assembled into submicrometer clusters containing relatively uniform mesoscale pores and then stabilized by coating with a thin layer of silica. Calcination of the materials at high temperatures connects the neighboring nanocrystals together and enhances the mechanical stability of the clusters and at the same time removes the organic surfactants and makes the TiO2 surface fully accessible to phosphopeptides. By coating the nanocrystal clusters with a layer of silica before calcination and removing it afterward through chemical etching, we have been able to make the cluster surface hydrophilic and negatively charged, thus enhancing the water dispersibility of the clusters and eventually their accessibility to phosphopeptides. The high selectivity and capacity of these mesoporous TiO2 clusters have been demonstrated by effectively enriching phosphopeptides from digests of phosphoprotein (alpha- or beta-casein), protein mixtures of beta-casein and bovine serum albumin, milk, and human serum samples. We also demonstrate that the self-assembly process brings the flexibility of incorporation of multiple components, such as superparamagnetic nanocrystals, to further facilitate the peptide separation.