Development Of Novel Enrichment And Identification Techniques For Phosphorylated/Glycosylated Proteins Based On Functionalized Materials

Proteomics aim at large-scale analysis of proteins in all biological objects, and it constitutes of expressional proteomics and functional proteomics. Investigation of genomics and proteomics gives us insights to the physiological, pathological and pharmacological processes of cells and organisms. However, there are extremely large amount of proteins with complicated properties, e.g. post-translation, protein-protein interaction, which bring great challenge to existing analytical methods. Therefore, it is necessary to develop new techniques and methodologies for better solution of proteomic research.Post-translational proteomics is one of the most important subjects in proteome research. Protein post-translational modification plays key role in many biological processes. There are more than 200 reported PTMs, among which phosphorylation and glycosylation are the most studied protein modifications. However, phosphorylated and glycosylated proteins are usually of low abundance in biological samples. It is difficult to detect these proteins in mass spectrometry without sample pre-treatment. Development of purification and enrichment methods for post-translational modified proteins are in great demand.The application of functionalized micro-/nano-particles in biomedical research area is gaining increasing attention due to there ease of manipulation and recovery during the past decades. These particles possess great specific surface areas and high surface activity. Functional modification based on these paricles can be easily performed. Up till now, functionalized materials are extensively applied in various biomedical applications such as cell separation, drug delivery, enzyme immobilization, and protein purification. With specific modification, functionalized materials can be effectively applied in phosphorylated/glycosylated protein separation and enrichment.Based on the proteome research background and the development trend of functionalized materials, the research interest of this work focused on the preparation of several kinds of functionalized materials and developing a series of techniques and methods to resolve current problems in the separation and concentration of phosphorylated and glycosylated proteins. The feasibility of these techniques and methods was validated with real biological samples. This dissertation is divided into four parts. In Chapter 1, advances in proteome research, current research techniques and methods of phosphorylated/glycosylated proteomics, and applications of functionalized materials in biological analysis were summarized in brief. The intention and meaning of this dissertation were explained.In Chapter 2, mesoporous TiO2 microspheres were synthesized by simple hydrothermal reaction, and successfully developed for phosphopeptides enrichment from both standard protein digestion and real biological sample such as rat brain tissue extract. The mesoporous TiO2 microspheres (the diameter size of about 1.0μm) obtained by simple hydrothermal method were found to have a specific surface area of 84.98 m2/g, which is much lager than smooth TiO2 microspheres with same size. The surface area of mesoporous TiO2 microspheres is almost two times of commercial TiO2 nano particle (a diameter of 90 nm). Both of these two TiO2 microspheres are successfully applied to selective enrichment of phosphopeptides generated from P-casein digest. However, when they are used for phosphopeptides enrichment from a complicated peptide mixture such as P-casein and BSA digest mixture, mesoporous TiO2 microspheres exhibit strong specific selectivity compared with amorphous TiO2 microspheres with smooth surface. When they are further used for phosphopeptide enrichment from rat brain tissue extract, the mesoporous TiO2 microspheres show the highest binding capacity as well as capture efficiency for phosphopeptides.223,47, 90 phosphopeptides were identified using mesoporous TiO2 microspheres, smooth TiO2 microspheres and commercial TiO2 nanoparticles, respectively. It has been demonstrated that mesoporous TiO2 microspheres have powerful potential for selective enrichment of phosphorylated peptides. Moreover, the preparation of the mesoporous TiO2 microspheres is easy, simple and low-cost. This mesoporous TiO2 material may be further used as affinity chromatography column packing material for comprehensive phosphorylated proteome research.In Chapter 3, an on-plate selective enrichment method is developed for fast and efficient glycopeptide investigation. Gold nanoparticles were first spotted and sintered on a MALDI-QIT-TOF-MS stainless steel plate, then modified with 4-mercaptophenylboronic acid. These spots were used to selectively capture glycopeptides from peptide mixtures, and the captured target peptides could be analyzed by MALDI MS simply by depositon of DHB matrix. Horseradish peroxidase was employed as a standard glycoprotein to investigate the enrichment efficiency.9 glycopeptides or glycopeptides fragments were identified with HRP concentration as low as 2.5×10-9 M. This on-plate glycopeptide enrichment strategy was further evaluated with other glycoproteins like fetuin, asialofetuin. It also succeeded at binding and identifying glycopeptides from a relatively complicated peptide mixture. The relatively small sample amount needed, low detection limit, and rapid selective enrichment have made this on-plate strategy promising for on-line enrichment of glycopeptides, which could be applied in high-throughput proteome research.In Chapter 4, Con A immobilized magnetic nanoparticles were synthesized for selective separation of glycoproteins. At first, a facile immobilization of Con A on aminophenylboronic acid-functionalized magnetic nanoparticles was performed by forming boronic acid-sugar-Con A bond in sandwich structure using methyl a-D-mannopyranoside as an intermedium. The selective capture ability of Con A modified magnetic nanoparticles for glycoproteins was tested using standard glycoproteins and cell lysate of human hepatocelluar carcinoma cell line 7703. In total 184 glycosylated sites were detected within 172 different glycopeptides corresponding to 101 glycoproteins. Also the regeneration of the protein-immobilized nanoparticles can easily be performed taking advantage of the reversible binding mechanism between boronic acid and sugar chain. The experiment results demonstrated that Con A modified magnetic nanoparticles by the facile and low-cost synthesis provided a convenient and efficient enrichment approach for glycoproteins, and are promising candidates for large scale glycoproteomic research in complicated biological samples.In summary, the main contributes of this dissertation is that we aimed at better solutions of post-translational modified proteome research, and developed several practical techniques for phosphorylated/glycosylated protein or peptide enrichment based on functionalized materials. According to the experiment results, these new techniques offer effective enrichment and identification of phosphorylated and glycosylated protein/peptide even in complicated biological samples, which demonstrated their powerful ability in post-translational proteome research.