Investigations On The Enrichment Of O-Glycosylatied Proteins Using Novel Thermosensitive Polymers

O-GlcNAc glycosylation is an important post-translational modification that widely exists in mamalian cells. It has similar structural characteristics and properties compared with protein phosphorylation and plays key roles in cell recognition, cell signaling, cellular immune responses, cell differentiation, gene transcription, cell metabolism and so on. Therefore a large scale identification of protein O-GlcNAc glycosylation is of great significance in studying the mechanism of disease occurrence and development. But there are several obstacles affecting the identification. O-GlcNAc glycosylation is low abundance in cells. The mass spectrometry signal of O-GlcNAc glycopeptides is suppressed by massive non-modified peptides. O-GlcNAc modification varies highly dynamically, and it is vulnerable to drop in preparation. O-GlcNAc modification is highly hydrophilic without any charges, thus making its response in mass spectrometry low. Neutral loss is vulnerable to occur in the secondary fragmentation and it is difficult to obtain MS/MS spectrum in high quality. In order to overcome these problems and enlarge the identification scale of O-GlcNAc modification, it is urgently needed to develop a fast, specific and highly efficient method to enrich and detect O-GlcNAc modification. This method is of great significance for revealing its function in biological and pathological processes.N-Isopropyl acrylamide (NIPA) is the most stable and widely used monomer for preparing temperature sensitive polymers. The dissolution of temperature sensitive polymers in aqueous solution changes with ambient temperature. It well dissolves in water and form homogeneous solution when the temperature is below its Lower Critical Solution Temperature (LCST). In contrast, when the temperature rises above its LCST, the polymer chains start crimping and exposing the hydrophobic groups in the polymer chains, which results in the formation of a heterogeneous solution and precipitation of the polymer. Low abundance proteins and peptides could be enriched through the fast transition between homogeneous solution and heterogeneous precipitation. Therefore, the inherent mass transfer resistance o: the conventional solid phase based enrichment matrix at the solid-liquid interface can be avoided by this temperature sensitive polymer. A homogeneous solution can be obtained foi faster and more efficient target molecule enrichment with reduced staric hindrance at temperature lower than LCST and the polymer can be easily recovered by rising the temperature to higher than LCST and centrifuging the precipitated polymer.In the first part, we have reviewed the status of the development of proteomics, the study of protein glycosylation, and the O-GlcNAc glycoproteins scale identification based on the mass spectrometry. O-GlcNAc glycoprotein enrichment methods commonly include lectin enrichment, antibody enrichment, metabolic labeling enrichment, chemical enzymatic markers enrichment,β-elimination-Michael addition of enrichment, hydrazide enrichment and so on. We also briefly introduce temperature-sensitive polymers and commonly conditions. We summarized the principles and applications of the Staudinger reaction.In the second part of this thesis, we have synthesized Staudinger reagent modified dextran microspheres. The standard O-GlcNAc modified proteins are enriched by a specific reaction between Staudinger groups and nitrine groups, which are labeled on the proteins. We have studied the dosage of microspheres and Staudinger reagent in preparing this enrichment material. Furthermore, we have enriched O-GlcNAc modified proteins from a mixture of bovine serum albumin and standard O-GlcNAc modified proteins labeled by nitrine groups. The results indicate that standard O-GlcNAc modified proteins labeled by nitrine groups can be specifically enriched by this material.In the third part, we have used free radical polymerization between NIPA and methyl acrylate to synthesize a linear polymer with ester groups under the initator potassium persulfate. The polymer with ester groups is hydrazided by hygrazine hygrate. The end of side chains of the polymer is modified by hydrazide groups. The hydrazidedtemperature sensitive polymer reacts with 2-(Diphenylphosphino)terephthalic acid 1-methyl 4-pentafluorophenyl diester phosphine-PFP ester. The temperature sensitive polymers with massive Staudinger groups on side chains are synthesized. The dispersity and temperature response properties help to enrich O-GlcNAc modified proteins and peptides in homogeneous system. The inherent mass transfer resistance of the conventional solid phase based enrichment matrix at the solid-liquid interface can be avoided by this temperature sensitive polymer, thus enhancing the enriching speed as well as efficiency. This method has been applied in the enrichment and identification of a mixture of bovine serum albumin and nitrine labeled O-GlcNAc modified bovine lens albumin.