A Novel Strategy For N-glycan Release Based On Analysis By Electrospray Ionization Mass Spectrometry

N-Glycosylation is one of the most important post-translation modifications (PTMs) of proteins and plays pivotal roles in many biological processes, such as immune response, signal transduction, cellular adhesion and tumor progression.Therefore, efficient release of sugar chains from skeleton protein is especially essential to the investigations in sugar chain structure and function.According to the bond between the sugar chain and protein backbone, sugar chain can be classified into N-glycans and O-glycans. For the release of O-glycans, chemical methods are usually employed, such as the conventional reductive β-elimination. In contrast, there are enzymatic methods for the intact release of N-glycans, such as glycosidase A (PNGase A) and glycosidase F (PNGase F). PNGase F is generally effective to all types of N-glycans except core al,3-fucosylated ones. For the release of N-glycans using PNGase A, proteolytic digestion of glycoproteins is required, because PNGase A does not act on intact glycoprotein and its enzymatic activity is low. Therefore, the development of new chemical methods for the release of N-glycans is important for high throughput preparation and analysis of N-glycans.This thesis developed a simple, rapid and cost-saving procedure allowing nonspecific release of N-glycans. The results are summarized as following:1. For the glycoproteins without core α1,3-fucosylation structures, N-glycans could be completely released in the non-protection manner by treatment with0.5M sodium hydroxide at50℃for16h. ESI-MS and MS/MS results show that the proposed method exhibits general applicability, high efficiency and high recovery. It permits quantitative release of N-glycans from Bovine Pancreas Ribonuclease B, Chicken Egg Albumin, Bovine Fetuin and Human Plasma under opitimized reaction conditions. The reducing N-glycans recovered by the method could be further tagged with various fluorescent reagents for LC, CE and LC-MS/MS analysis.2. For the samples containing core al,3-fucosylated structures, N-glycans could be completely released and dervatized in situ with PMP (1-phenyl-3-methyl-5-pyrazolone) in the protection manner by treatment with0.5M aqueous NaOH solution containing0.7M PMP in75℃for32hours. It permits quantitative release of N-glycans from Horseradish Peroxidase, Honeybee Venom Phospholipase A2, Fagopyrum esculentum Moench Pollen, Arabidopsis Thaliana Leaves and Nicotiana tabacum L. Leaves under opitimized reaction conditions. ESI-MS/MS and MS/MS analysis showed that PMP greatly diminished "peeling" reaction of core α1,3-fucosylated N-glycans. It is extensively applicable to the samples containing core α1,3-fucose in plants and insects. In addition, the released N-glycans as fluorescent PMP derivatives not only exhibit improved detection sensitivity in mass spectra, but also can be directly subjected to analysis by LC, CE and LC-MS/MS.