Development And Application Of Stable Isotope-Labeled Receptors

As a ubiquitous post-translational modification, glycosylation has an important impact on the structure and function of proteins. Studies demonstrate that many diseases are associated with structure of N-sugar chain and changes in expression of protein, by means of quantitative analysising of N-sugar chain can find the sugar chain disease abnormal changes occur accurately, and the method plays an invaluable role with diagnosis and treatment of the disease. Currently MS detection based on the chemical tag is a sugar chain by N-effective quantitative methods, but these techniques would have carried out additional chemical reactions in the experiment and the effects of marking have an adverse impact on sample purification. Endo-M enzymatic transglycosylation reaction is excellent solution to above problems. The enzyme can cut N-sugar chains, and the same time transfer sugar chain to the glycosyl acceptor.In this study, to provide the necessary sugar chains of the receptor for the establishment of a sugar chain on the N-transferase quantitative methods, several stable isotope-labeled receptors which could improve detection sensitivity and resolution MS were synthesized.In chapter two, according to (do/d8)-PDPZ-Boc-Asn-GlcNAc of stable isotope labeled receptors, utilizing the Endo-M-N175Q sugar chain transfer reaction, and asialoglycoprotein peptide SGP sugar chain was transfered. By UPLC-ESI-TOF-MS analysising, with detecting do-/d8-receptors labeled transglycosylation products, and their relative abundance ratio close to1:1. This result, which establish a kind of simple operation, high sensitivity, high accuracy of the N-sugar chain relative quantitative analysis method, lays a chief foundation.In the chapter one, the two pairs of light and heavy stable isotope labeling Endo-M enzymatic glycosylation acceptor and two deuterated glycosylated receptors were synthesized, the optimal conditions of single reaction was probed, the purification method was optimized, all compounds were characterized by NMR, MS, UV, LC, melting point. By means of N-carbohydrate sugar chains for relative quantitative analysis researching, to provide the needs of derivatization reagent.In the chapter two,the stable isotope-labeled receptors (do/d8)-PDPZ-Boc- Asn-GlcNAc and sialicacid glycopeptide SGP were transfected glycosylation by UPLC-ESI-TOF-MS detection. Through the comparison of the (do/d8)-transglycosylation product relative abundance, and relative quantifying different samples of sugar chains, to establish a kind of relative quantitative analysis methods which simple operation, high sensitivity, high accuracy.In the chapter three, new receptors can be combined with a carboxylic acid synthesized and has a UV absorption, it can be detected by HPLC ursodeoxycholic acid, chenodeoxycholic acid and other bile acid substances derivatized products, new subject body water solubility, high mass response by MALDI-TOF-MS detection of carboxylic acids such as benzoic acid derivative of the product. To establish a less efficient and simple RP-HPLC analysis.In the chapter four, the classification of sugar chains and their biological functions was introduced, the current technology of modern biological mass spectrometry quantitative analysis of sugar chains were summarized.