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.
The project addresses the problem of the development of new analytical methods for the enrichment of phosphopeptides from complex biological matrixes. During enrichment the supports to metal cations and metal oxides are not inert, since they affect, with unspecific interactions, the binding of interfering non-phosphorylated peptides. However, most of the new phases are developed and employed for enrichment with simple mixtures and direct MALDI analysis. In this general context one must separate the proof of concept from the final applications. In fact, if the direct MALDI analysis is a very convenient technique suitable for peptide samples of limited complexity, still the new materials and analytical methods will be applied to extremely complex samples, such cell and tissue extracts. Naturally methods must be integrated in analytical workflows as close as possible to the ones employed in phosphoproteomics research, which at least comprise a monodimensional chromatographic separation, needed for the analysis of complex peptide mixtures. These issues will be dealt with in the present research project, thus the main innovations are the following:
1- For IMAC method development, a new composite material suitable for the batch magnetic solid phase extraction of phosphopeptides will be prepared. In order to immobilize Ti4+ ions on the surface of the magnetite nanoparticles, they will be first covered by a silica shell and then modified to expose at the surface bromine containing groups. Glycidyl methacrylate will be subsequently polymerized from these groups using the ¿grafting from¿ approach by the activator regenerated by electron transfer atom transfer radical polymerization technique. Finally, the glycidyl groups will be reacted with iminodiacetic acid to functionalize the material with moieties suitable for coordination. Glycidyl methacrylate was chosen as starting polymer for this class of Ti-IMAC materials since, other than previously exploited polymers, it has a hydrophilic character which could help minimize unspecific binding due to hydrophobic interactions. However, different polymers and ligands will also be tested for method improvement and comparison.
2- The enrichment protocol will be optimized with a real biological complex sample by means of nanoHPLC coupled to high resolution tandem mass spectrometry. This is a key feature of the proposed project. In fact, in the literature, tests on real complex matrices, such as whole cell extracts, are usually omitted but they represent the final application of such enrichment methods. Moreover, results are frequently obtained by direct MALDI analysis, which is not suitable for complex samples or current shotgun proteomics analysis. Therefore, such results are at proof-of-concept level and need further application for clear assessing the scope of the new proposed method. In this context, for the presented research project yeast protein digests will be employed for optimization of the enrichment protocol and results will be assessed by nanoHPLC-MS/MS. This would not only ensure compatibility of the developed enrichment method with the final up-to-date method for protein analysis, but also it allows to clearly evaluate the potential of the new method with a real sample and compare it to established systems.
3- The type of extraction method which will be exploited is based on magnetic separation. In most phosphoproteomics applications the stationary phases are packed, commercially or manually, into pipette tips, later employed as miniaturized spin columns. Such devices are easy to use; however, it is difficult to scale them up for large experiments. A batch format would overcome such difficulties by simple increase of the amount of sample to be enriched and of the phase employed for enrichment. These advantages will be exploited for analysis of real matrices, in particular biofluids, to demonstrate the superiority of this approach over commercial ones.
4- Application to samples of clinical importance will be the final goal of the present research project. In particular, the materials will be applied to the enrichment of endogenous peptides in serum and saliva. Such biofluids are currently employed for clinical analysis, however methods for the analysis of their endogenous peptides are currently lacking. Nevertheless, the potential of such peptides could be important, since they can originate from pathological processes and thus could serve as biomarkers for future diagnostic applications. In this context, the development of an analytical method based on shotgun peptidomics would positively impact on both the search for new biomarkers and the development of reference methods for their analysis. This will be pursued employing the new Ti-IMAC materials developed within the project and carbonaceous materials developed in this laboratory.
The proposed study would increase knowledge in the field of shotgun phosphoproteomics and peptidomics.