Relative Quantification Of N-Linked Glycan Based On Endoglycosidase

Glycosylation is known as an important process in many biological events, such as, cell adhesion, host-pathogen interactions, quality control of proteins, and retention of in vivo activities of many cytokines. Studies demonstrate that many diseases are associated with structure of N-sugar chain and changes in expression of protein, such as cancer, metabolic disease and neurodegenerative diseases. By monitoring the change of N-sugar chain will help to understand and control the process of illness, which takes great significance in the diagnosis and treatment of the disease. By the use of the enzymatic transglycosylation of Endo-M combined with stable isotope labeling based on mass spectrometry, we can make the relative quantitation of the N-sugar chains come true without making an adverse impact on sample purification.In chapter one, we introduced the classification and biological functions of the sugar chains firstly. And then, we briefly summarized the methods for the extraction and separation of sugar chains and the current technology for the analysis of sugar chains.In chapter two, we improved the method for the synthesis of do/d8-PEPZ-Boc-Asn-GlcNAc on the last step. By the use of DMT-MM and methanol system instead of TPP-DPDS as catalyst, the yield was increased from 40% to more than 80%. In addition, the reaction solvent in the synthesis of do/d8-MPEPZ-Boc-Asn-GlcNAc was replaced by acetonitrile-DMF instead of methanol, so that the reaction would be able to finish completely by once. The products were characterized by NMR, IR, UV, MS, LC.In chapter three, on the basis of LC-ESI-MS analysis, we compared the reaction yield of four lightly labeled receptors PEPZ-Boc-Asn-GlcNAc、PDPZ-Boc-Asn-GlcNA、MPEPZ-Boc-Asn- GlcNAc、MPDPZ-Boc-Asn-GlcNAc made by our laboratory with SGP in the presence of Endo-M-N175Q, and finally selected PEPZ-Boc-Asn-GlcNAc as the best one. Then we determined the best dosage of enzyme as 5 mU, and the optimal reaction time as 3 hours. Furthermore, the linearity and dynamic range of this stable isotope labeling method were evaluated.