New Method To Glycan Structure Analysis Of The Glycoproteins Using Chromatography And Mass Spectrometry

Glycosylation is one of the most important post-translational modifications, and it plays important roles in many physiological processes. The relationship between the disease and the varying pattern and abundance of glycosylation has been found, therefore, the analysis of the structure of saccharide has been very important in proteomics. Characteristics of the saccharide including complexity, diversity, micro-heterogeneity and limited quantities from organism bring great challenge to present analytical methods. So it is necessary to develop new techniques and methodologies for the analysis of the structure of saccharide.The research interests of this work focused on the analysis of the structure of saccharide and the new technique for the rapid digestion of glycoproteins. We established a high sensitive capillary HPLC system coupled with laser induced fluorescence detector (LIF). A new fluorescent reagent was used in labeling the reducing end of saccharides for the first time. A novel method for the study of the structure of saccharide was proposed and applied. Based on the hydrophilic monolithic enzymatic microreactor, the rapid glycoprotein digestion was improved. The research work in this thesis was divided into five chapters.In chapter 1, advances in glycosylated proteomics was summarized. Current research techniques and methods of the analysis of the structure of saccharide and the digestion of glycoprotein were described in detail. The intention and meaning of this dissertation were illustrated.In chapter 2, the method of controlled hydrolysis of oligosaccharide was established. In order to obtain detailed structural analysis of oligosaccharide, the oligosaccharide was degraded to smaller carbohydrate that could be used in conjunction with instrumental analyses. Acid-catalyzed controlled hydrolysis was one of the chemical methods for degrading oligosaccharide. By this method, the smaller oligomers with new reducing ends were formed. The generation of a reducing end ladder provided a means for subsequent derivations with labeling reagent. It was meaningful to develop new method for analysis of the structure of the saccharide. In this chapter, the oligosaccharides of maltoheptaose and P-cyclodextrin were employed to investigate the conditions of acid hydrolysis including different acids, heating methods, etc., and these conditions had been evaluated and optimized. The controlled hydrolyzed products were determined by MALDI-TOF MS. The conditions of controlled hydrolysis and the composition of controlled hydrolyzed products were determined. Finally, we successfully established the approach of controlled hydrolysis of oligosaccharide which was an important basis for the analysis of the structure of oligosaccharides.In chapter 3, we established a high sensitive capillary HPLC system coupled with laser induced fluorescence detector (LIF) and developed a new method for the analysis of the structure of saccharide. The limited amount of saccharide from organism was a great challenge to the analysis of the structure of saccharide. Therefore, it was necessary to develop a new method for the analysis of low levels of sample. We set up a high sensitive capillary HPLC-LIF system including conventional pump system, homemade capillary column, and laser induced fluorescence detector (LIF). This system provided the possibility for the realization of the micro-analysis of sugar chain. In general, saccharide in their native state was poorly detected in chromatography and MS. In order to improve the detection of saccharide, labeling method was usually used. For the purpose that the saccharide could be detected in this new system, maltoheptaose was employed as model oligosaccharide to select the suitable labeling reagent. A variety of derivative reagents was used and the characteristics of different reactions were discussed. Finally, the high sensitive fluorescent reagent was selected as the target and firstly used in labeling reducing oligosaccharides. The labeling reaction conditions were optimized, and a new method for analyzing the structure of saccharide was developed, which was combined with partial acid hydrolysis, fluorescent labeling, a high-sensitivity capillary HPLC-LIF system and MS. This approach provided a possibility for the analysis of complicated saccharides in glycoprotein.In chapter 4, we developed a new method for purifying saccharides from the mixture of protein and saccharides. At the same time, the new approach to the analysis of saccharides from glycoprotein was established. The varying pattern and abundance of glycosylation could effect on the physiological functions, therefore, it was very important to study the saccharides in glycoprotein. We advanced a new method for the analysis of saccharides from glycoprotein based on the study of oligosaccharide. RNase B was used as a model glycoprotein, and saccharides were obtained by glycoprotein digestion with PNGase F. To get the sugar from the mixture, purification was necessary. The different purifying methods were used, including C18 SPE column, C18 ZipTip and homemade C18 capillary column. It was found that the homemade column shown well retention for protein and was suitable for purifying the micro-sample with its small volume. So the purifying method was set up. Subsequently, we built the new method for the analysis of saccharides from glycoprotein, and this method combined with partial acid hydrolysis, fluorescent labeling, a high-sensitivity capillary HPLC-LIF system and MS. A fraction of labeling saccharides from glycoprotein was used as a model in the research, and the satisfactory results were achieved.In chapter 5, a technique for the rapid digestion of glycoprotein was improved. The on-line digestion system was established and successfully applied for the analysis of several glycoproteins. Compared with the conventional in-solution digestion of protein, immobilized enzyme has been widely utilized owing to its advantages of higher enzyme concentrations in it that lead to shorter digestion time and high efficiency. Our group had developed a simple and efficient method for the synthesis of monolithic enzymatic microreactor with hydrophilic substances. In this study, we extended the application of the monolithic enzymatic microreactor in the proteolysis of glycoprotein because of its good hydrophilicity which could avoid non-specific hydrophobic interactions and make the transfer of glycopeptides easier. Using HRP as a model glycoprotein that detected by MALDI-QIT-TOF MS after digestion, high performance of the monolithic enzymatic microreactor was demonstrated. Compared with the conventional in-solution digestion, in-microreactor digestion needed the less time. In addition, the on-line monolithic enzymatic microreactor-based μHPLC analysis system was built, which is utilized in the rapid digestion and separation of glycoproteins. Furthermore, we made progress in the rapid and efficient glycoprotein digestion by microwave-assisted microreactor digestion. After several experiments under microwave-assisted condition, the performance of the microreactor was demonstrated to be fine. These methods extended applications of the hydrophilic microreactor in glycoprotein digestion.In summary, this thesis focuses on the key and challenging problems of glycosylated proteomics. We advanced a new approach for the analysis of the structure of oligosaccharides. Partial acid hydrolysis was used in this method, and the hydrolysis conditions were discussed. In order to micro-analyze oligosaccharides, a capillary HPLC-LIF system was set up. A high sensitive fluorescent reagent was selected for labeling the saccharides and this reagent was firstly used in the labeling the reducing end of oligosaccharides. Furthermore, we made progress in the rapid digestion of glycoprotein. The on-line digestion and micro-HPLC system was established and successfully applied to the analysis of several glycoproteins. Both of them provided an important foundation to realize the analysis of complicated saccharides in glycoproteins and the rapid digestion of glycoproteins on a large scale.