A New Method For Separation And Analysis Of Phosphorylated Proteins Based On Novel Functionalized Nanomaterials

Protein phosphorylation is one of the most important post-translational modifications, affecting both the folding and function of proteins. It is known to be involved in the regulation of diverse processes including cellular signaling and communication, cell differentiation and survival and it has also been vividly described as the molecular switch of the cellular activities. Therefore, to locate the exact residues that are phosphorylated and investigate the level of phosphorylation for individual sites on a given protein is of paramount importance. These dates can in principle provide insight into normal cell biology, and can make a fundamental understanding of the biological processes controlled by protein reversible phosphorylation at the molecular level. Mass spectrometry has emerged as a reliable and sensitive method for the localization of protein phosphorylation sites and is the favored method for phosphorylation analysis of signaling proteins. Unfortunately, due to substoichiometric phosphorylation and the serious ion suppression, the signals of the phosphopeptides are often suppressed by nonphosphorylated peptide residues contained in protein digests, which means that satisfactory results cannot be obtained by direct mass spectrometry analysis of protein digest. Therefore, MS combined with enrichment strategies for phosphorylated proteins and peptides is the tool of choice for the identification of novel phosphorylation sites. New technologies for separation and enrichment of phosphopeptides from protein digest mixtures is in urgent demand.Recently, functionalized namomaterials have attracted much attention because of their unique performance in mechanical, electrical, magnetic, optical, thermal, etc and have been widely applied to numerous fields, including sensors, semiconductor, catalyst, biological, medical, household appliances, environmental protection, coatings and mechanical. As new analysis techniques, functionalized namomaterials have also been widely used in the study of proteomics.In this study, we focused on preparing several different kinds of novel functionalized namomaterials with large surface area and using them for selective enrichment of phosphopeptides. This dissertation is divided into five parts.In Chapter1, we frist summarized the art of phosphoproteomics in details. This review gives an overview over the most frequently used methods in isolation and detection of phosphoproteins and phosphopeptides such as specific enrichment or separation strategies as well as the localization of the phosphorylated residues by various mass spectrometric techniques. At last, the application of the functionalized namomaterials in proteomics has been summarized.In Chapter2, we synthesized Fe3O4@mesoporous TiO2magnetic microspheres with core-shell structure and large surface area for selective enrichment of phosphopeptides. We used them to isolation and enrichment of the phosphopeptides from tryptic digestion of both standard proteins and real samples. Due to that the as-made Fe3O4@mesoporous TiO2microspheres have large surface area, good dispersivity and biocompatibility, they have been demonstrated as a powerful tool for phosphoproteomics research.In Chapter3, TiO2/graphene composites were synthesized through a simple one-step hydrothermal reaction and used to selectively capture phosphopeptides from pepetide mixtures. Graphene has attracted a great deal of interest recently because of its outstanding mechanical, electrical, thermal, optical properties and theoretically high surface area of～2600m2g-1. Due to their tendency to aggregate when graphene dispersion solutions are dried, graphene decorated with metal nanoparticles and metal oxides nanospheres have recently been reported. Owing to the excellent property of TiO2for enrichment of phosphopeptides, we synthesized TiO2/graphene composites and used them to selectively capture phosphopeptides. Finally, we have demonstrated that TiO2/graphene composites exhibit high sensitivity and selectivity to phosphopeptides.In Chapter4, on the basis of work described in the third chapter, magnetic FesO4were introduced in the composites and Fe3O4/graphene/TiO2composites were prepared. Besides that the specificity adsorption ability of titanium dioxide to phosphopeptides and large specific surface area of graphene were retaining, the composites was given magnetism, which makes the separation the enrichment process easier, quicker and more convenient. In addition, we applied the composites for the enrichment of phosphopeptides from tryptic digest of human liver cancer tissue.In Chapter5, a new plate-based technique for fast enrichment of phosphopeptides is described. MALDI target plate is covered with our new synthesized alumina hollow spheres and analyzed by MALDI-TOF-MS after enriching phosphopeptides from peptide mixtures. Owning to the special hollow structure, a higher surface area was obtained which makes it highly efficient for desired phosphopeptides. As a result, this enrichment approach has proven to be effective and sensitive for analysis of phosphopeptides from peptides mixtures and provides a promising way to establish online investigation strategy for phosphopeptides of biological samples.In summary, the main contribution of this dissertation is that we initially synthesized several different kinds of functionalized nanomaterials with large surface area and successfully utilized them for phosphopeptides enrichment.