Development Of Novel Thermo-responsive Polymers For Sensitive Detection,Efficient Enrichment And Identification Of O-GlcNAc-modified Proteins

O-linked ?-N-acetylglucosamine(O-GlcNAc)is a ubiquitous intracellular post-translational modification in living cells.Despite their extremely low-abundance,O-GlcNAc-modified proteins play important roles in vertebrates cells,from signal transdution and transcription to gene regulation and cell cycle control.The aberrant O-GlcNAc modification in proteins is found correlated with many major human diseases such as psychological disease,metabolizable disease and cancer.Due to its extremely low stoichiometry,it is particularly challenging to detect and identify O-GlcNAc proteins in complex biological sample.In order to achieve ultra-sensitive detection of O-GlcNAc-modified proteins,we developed a new "chemical antibody" based on Western Blot method using functionalized temperature-sensitive polymers.Furthermore,it is necessary to enrich the low abundant O-GlcNAc protein before mass spectrometry analysis to achieved sensitive identification.However,the enrichment efficiency is limited using conventional solid/insoluble enrichment materials due to the solid-liquid interface mass transfer resistance and steric hindrance between the enrichment materials and target molecules in the solution.Therefore,in this work we designed and synthesized a novel type of functional temperature-sensitive polymer as a convenient tool for enrichment of O-GlcNAc-modified proteins.The main contents are as follows:The first chapter summarizes proteomics,O-GlcNAc proteins and the corresponding enrichment methods,staudinger ligation,temperature-sensitive polymer and Western Blot.In the second chapter,we developed a "chemical antibody" based on Western Blot technique using functionalized temperature-sensitive polymers.Ultra-sensitive detection of the O-GlcNAc-modified proteins from HeLa cells was achieved.N-Isopropyl acrylamide,acrylic acid and initiator Br?P-CD were used for radical polymerization.The resulting polymer was subjected to heavy bifunctional modification with triarylphosphine and horseradish peroxidase to obtain a "chemical antibody" that specificly recognizes the azide labeled protein.The temperature-sensitive polymer was covalently coupled to the O-GlcNAc modified protein via Staudinger ligation between azide and triarylphosphine.The chemiluminescence signal was significantly amplified by the high loading of immobilized HRP.Thus ultra-sensitive detection of O-GlcNAc modified protein using "chemical antibody" based on Western Blot technique was achieved.Furthermore,the polymer can be separated and recovered by centrifugation after heating via exploiting its own temperature responsive characteristic.Therefore,recycling and reuse of the bifunctional polymer was realized.The maximum HRP loading capability of this polymer was 85.6711.17?g mg-1.The detection sensitivity of this method was 0.5?g total protein from HeLa cells.Compared with conventional Westen Blot method using antibody,the temperature-sensitive polymer based on"chemical antibody" has improved specificity and reduced false positive rate.The application of this "chemical antibody" can be expended to detect other azide labled protiens by Western Blot demonstrating the strong potential of this new method.In the third chapter,we designed and synthesized a novel type of triarylphosphine-modified temperature-sensitive polymer as a convenient tool for enrichment of standard O-GlcNAc-modified proteins.N-Isopropyl acrylamide and methyl acrylate were used as the monomer and potassium persulfate as the initiator for conventional free radical polymerization.Next,the obtained polymer was treated with hydrazine hydrate and 2-(Diphenylphosphino)terephthalic acid 1-methyl 4-pentafluorophenyl diester to produce triarylphosphine-modified temperature-sensitive polymer.The functional temperature-sensitive polymer was characterized by gel permeation chromatography(GPC),dynamic light scattering(DLS)and X-ray photoelectron spectroscopy(XPS).The temperature-sensitive polymer can be used to highly selective capture the low abundance azide-labeled O-GlcNAc protein in the sample via covalent conjugation between azide and triarylphosphine.By exploiting its temperature response properties,the solubility of the temperature-sensitive polymer in aqueous solution can be easily controlled by adjusting the environmental temperature.The polymer was fully dissolved during enrichment and precipitated for separation and recovery,therefore homogeneous enrichment and heterogeneous separation was achieved.It can obviously reduce mass transfer resistance and steric hindrance in the enrichment.The polymer was heavily functionalized with enrichment groups which led to increased the collision probability between the enrichment materials and the O-GlcNAc modified proteins.The enrichment efficiency was verified by mass spectrometry analysis.It was successfully applied in the efficient enrichment of standard O-GlcNAc proteins from protein mixture containing large amount of BSA.