Ricerc@Sapienza

Protein phosphorylation is one of the most important post-translational modifications, by means of which living organisms regulate cell activities; the knowledge of the phosphorylation state provides valuable information, useful to elucidate disease mechanisms and plant metabolism. Since phosphoproteins and endogenous phosphopeptides are expressed in low abundance, suitable systems for their enrichment are needed. However, challenges do still exist, and available systems need further improvement, especially for real complex samples and for shotgun phosphoproteomics analysis. In this context, nanomaterials coupled with magnetic solid-phase extraction provide interesting sorbents, with the possibility of enriching the target compounds directly in the sample solution and concentrate them by application of an external magnetic field.
In this regard, the aim of the project will be the development of new magnetic materials for the enrichment of phosphopeptides, either after protein isolation and digestion or targeting the endogenous peptides present in the matrix. Fe3O4 nanoparticles will be produced and derivitized with a hydrophilic polymer, such as. polyglycidyl methacrylate, in order to reduce unspecific binding driven by hydrophobic interactions; suitable chelators, such as iminodiacetic moieties, will be covalently bound for Ti4+ or other cation immobilization. Alternatively, materials coupling magnetite with the large surface area of carbonaceous materials and metal oxides will also be prepared. Such materials will be tested for phosphopeptide enrichment in complex real matrices, such as yeast extracts, and finally applied to samples where the investigation of endogenous peptides can provide a method for identification of new biomarkers, such as the case of serum and saliva. The enrichment protocol will be embedded within a typical shotgun proteomics or peptidomics workflow, to move from the proof of principle level to the real world application one.