Preparation And Application Of Materials To The Enrichment Of Phosphopeptides

Phosphorylation of protein is the addition of a phosphate group to a protein under the regulation of kinases. This process is reversible, and phosphoproteins can be dephosphorylated by phosphokinases. Reversible phosphorylation of proteins is not only closely related to signal transduction pathways, but also involved in many cellular processes, such as proliferation, differentiation and apoptosis. In particular, simultaneous phosphorylation of adjacent amino acids will greatly affect the biological activity of the proteins. Now, mass spectrometry based techniques have become the foremost choice for phosphoproteomics analysis, because of their high sensitivity and accuracy. However, due to their low abundance, substoichiometry and low ionization efficiency in mass spectrometry, it remains difficult to analyze phosphopeptides, especially multi-phosphopeptides and endogenous phosphopeptides. Therefore, isolation and enrichment of phosphopeptides are essential before MS analysis. In this dissertation, by combining novel technology and theory with the enrichment methods of phosphopeptides, we develop several approaches for the separation of phosphopeptides, multi-phosphopeptides and endogenous phosphopeptides.1. The development of phosphoproteomics analysis is reviewed. It emphasizes on the enrichment methods of phosphopeptides and the application of novel technology in these methods.2. A novel strategy for the enrichment of phosphopeptides based on the particular affinity of zirconium arsenate towards the phosphate group was developed. The nanoscale material not only provides large specific surface area for binding target species, but can also be easily dispersed in solution. The magnetic property of the magnetic nanoparticles was convenient for the enrichment of phosphopeptides from a complex mixture. The excellent performance of this material was demonstrated by its specific capture of phosphopeptides from both the tryptic digest of standard phosphoproteins and human serum.3. Various kinds of perovskites were compared for their selective enrichment of phosphopeptides. Perovskites (MgTiO3, CaTiO3, SrTiO3, BaTiO3and CaZrOs) as well as metal oxides showed high affinity for capturing phosphopeptides, but they displayed higher selectivity than Z1O2and TiO2. Among the perovskites, CaTiO3and SrTiO3displayed better selectivity for phosphopeptides than MgTiO3, BaTiO3and CaZrO3, especially CaTiO3having the best specificity. The different selectivity between metal oxides and perovskites could be due to their unique crystal structures and electrophilic abilities. Finally, we compared the performance of CaTiO3and TiO2for the enrichment of phosphopeptides form tryptic digest of Jurkat-T cell lysate, and CaTiO3demonstrated very high specificity and selectivity for phosphopeptides in the complex biological sample.4. A highly selective enrichment method for the enrichment of phosphopeptides or multi-phosphopeptides based on phosphonate-modified metal oxides was developed. Various compounds with different numbers of phosphate group were adsorbed on the surface of ZrO2and TiO2to obtain phosphonate-modified metal oxides. Among them, phosphoric acid modified metal oxides (1P-ZrO2and1P-TiO2) could significantly enhance the binding selectivity towards phosphopeptides; and alendronate-modified metal oxides (2P-ZrO2and2P-TiO2) showed highly selectivity for the enrichment of multi-phosphopeptides. In addition, the detection sensitivity was greatly improved while using these novel materials. The mechanism of the specific enrichment of phosphopeptides or multi-phosphopeptides by phosphonate-modified metal oxides was proposed to ligand exchange and blocking of strong adsorption sites by the compounds containing phosphate group. Finally, ZrO2, lP-ZrO2and2P-ZrO2were applied for the enrichment of phosphopeptides form tryptic digest of Jurkat-T cell lysate.5. Titanium grafted magnetic mesoporous silica (Fe3O4@Ti-mSiO2) was developed and applied for the enrichment of endogenous phosphopeptides. In this material, titanium locates on the surface of mesoporous silica, which can provide abundant adsorption sites for phosphopeptides. In addition, coating a thin layer of SiC>2and TiO2composite does not change the pore size of ordered mesoporous silica, which is beneficial for extracting endogenous phosphopeptides and excluding high-MW proteins. Moreover, the magnetic property will provide us with simple, fast and effective isolation of phosphopeptides from complex mixture. The prepared Fe3O4@Ti-mSiO2particles can be successfully applied for the enrichment of endogenous phosphopeptides from human serum.6. By a sol-gel method, titanium-containing magnetic mesoporous silica sphere was synthesized and applied for effective enrichment of endogenous peptides and phosphopeptides from both tryptic digests of standard protein and human serum. Mesoporous silica spheres have been successfully applied for the enrichment of endogenous peptides, therefore, via combination of size-exclusion mechanism, hydrophobic interaction and affinity chromatography, titanium-containing magnetic mesoporous silica sphere was successfully applied to simultaneously extract and separate endogenous non-phosphopeptides and phosphopeptides. For the limited amount of biological samples, simultaneous enrichment and separation of endogenous peptides and phosphopeptides is highly important.