Based On The Enrichment Of Phosphorylated Peptides And Glycopeptides Of New Functional Magnetic Nanomaterials

Protein phosphorylation is one of the most important post-translational modifications.Protein phosphorylation, which has also been vividly described as the molecular switch of the cellular activities, is known to be involved in the regulation of diverse processes including cellular signaling and communication, cell differentiation and survival.It is therefore important to locate the exact residues that are phosphorylated and investigate the level of phosphorylation for individual sites on a given protein. 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, 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.And new technologies for selective enrichment of phosphopeptides are in great demand.Meanwhile, glycosylation is one of the most important and abundant post-translational modification in nature. Glycoproteins play important roles during molecular and cellular recognition in development, growth, and cellular communication and in particular are involved in cancer progression and immune responses. Glycoproteins have been used as therapeutic targets and biomarkers for cancer prognosis, diagnosis,and monitoring. The determination of these glycoproteins is still an enduring analytical challenge with current ionization technologies such as matrix-assisted laser desorption/ionization and electrospray ionization. To solve this problem, it is necessary to have an enrichment step of glycopeptides to enhance the detection sensitivity to low-abundant but multi-glycosylated proteins prior to MS analysis.Additionally, functionalized magnetic microspheres are the most commonly selected substrates as affinity probes because of the ease of isolation of the magnetic-material-target conjugate from the sample solution based on their magnetic properties, and have been widely applied for the proteomics research. In this study, we focused on preparing several different kinds of novel functionalized magnetic microspheres which used for phosphopeptides and glycopeptides enrichment. This dissertation is divided into five parts.In Chapter 1,state of the art in phosphoproteomics is summarized 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.Meanwhile, the author states the different strategies for the glycopeptides enrichment. At last, the application of the functionalized magnetic microspheres for bio-separation has been summarized.In Chapter 2, we developed phosphate functionalized magnetic Fe3O4@C microspheres to immobilize Zr4+ ions for selective extraction and concentration of phosphopeptides for mass spectrometry analysis.The Fe3O4@C microspheres were functionalized with phosphate groups through a simple hydrolysis reaction using 3-(trihydroxysilyl)propyl methylphosphate. And the Zr4+ ions were immobilized on phosphate functionalized magnetic microspheres by using phosphate chelator. Finally, we successfully employed Zr4+-phosphate functionalized magnetic microspheres to selectively isolate the phosphopeptides from tryptic digests of standard protein and real samples including rat brain. All the experimental results demonstrate the enrichment efficiency and selectivity of the method we reported here.In Chapter 3,we synthesized Fe3O4@C@Ta2O5 magnetic microspheres and applied them for phosphopeptides enrichment. Recently, niobium pentoxide was proved to have the ability for selective enrichment of phosphopeptides.Considering the proximity of tantalum to niobium, we supposed that Ta2O5 can be used as affinity probes for phosphopeptides enrichment. To demonstrate its ability for selective enrichment of phosphopeptides, we applied Fe3O4@C@Ta2O5 magnetic microspheres to isolation and enrichment of the phosphopeptides from tryptic digestion of standard proteins and real samples.In Chapter 4, we synthesized core-shell Fe3O4@C@SnO2 magnetic microspheres. The Fe3O4@C@SnO2 microspheres were applied to enrich phosphopeptides for mass spectrometry analysis.Different from titanium and zirconium, regular sol-gel process is not effective in generating tin dioxide on the magnetite particles because the tin oxide tends to crystallize during the reaction. Herein, we reported a simple but effective method combining solvothermal and hydrothermal reactions to synthesize Fe3O4@C@SnO2 microspheres.We have also demonstrated that Fe3O4@C@SnO2 microspheres are the promising materials for convenient, efficient enrichment of phosphopeptides.In Chapter 5,we reported an alternative strategy to synthesis core-shell structure Fe3O4@C@Au magnetic microspheres by a self-assembly approach. The as-synthesized Fe3O4@C@Au magnetic microspheres were functionalized with 4-mercaptophenylboronic acid and successfully applied for selective enrichment of glycopeptides and glycoproteins.At first, the Fe3O4@C magnetic microspheres were synthesized by the two step reactions including solvothermal and hydrothermal reactions.Then, Fe3O4@C@Au magnetic microspheres with core-shell structure were obtained by a self-assembly approach. Finally, the Fe3O4@C@Au magnetic microspheres were modified with 4-mercaptophenylboronic acid. The 4-mercaptophenylboronic acid-modified Fe3O4@C@Au magnetic microspheres were successfully applied to selective enrichment of glycoproteins and glycopeptides.In summary, the main contributes of this dissertation is that we initially synthesized several different functional magnetic materials and successfully utilized them for phosphopeptides enrichment.