Analysis Of Antigenic Determinant Glycans Of Peanut Allergy Glycoprotein Ara H1 By Mass Spectrometry

Food allergy has been considered as one of most unhealthy factor and affords plenty of digestive symptoms such as abdominal pain, vomiting and diarrhea to anaphylactic shock, skin problems, asthma and other symptoms even death. Peanut is a kind of common food allergens and represents a threat for sensitive people in worldwide especially for children.Compared to other allergens, peanut shows other feature, that very few people will has tolerance against allergen when they grow up and for most people especially children possess the life-time susceptibility. Currently, the reports about peanut allergen are mainly focused on allergic protein. Thus, the systematical analysis of glycan structure on allergen protein is remains lacking and not complete.Therefore, In order to further study the glycan structure on the allergic protein, this study chose the total protein of peanut seeds and the main allergen glycoprotein Ara h1 as the experimental materials, used a new method which developed by our laboratory, called "one pot" to release the glycans on target protein and labled it with PMP. Then, the glycans were purified by C18 and graphitic carbon columns. The ESI-MS, MS" and HILIC-MS methods were used to analysis and identify the structure of these glycans. The study provides the useful information for the later study of the peanut allergen glycans biological function research. The experiment conclusions are as follows:1. The identification of SDS-PAGE show the glycoprotein of Ara h1 is homogeneous and its molecular weight is about 63.5 kD.2. The Bradford and Phenol-sulfuric acid results show that the protein content of allergen Ara h1 is 95.28% and sugar content is 2.07%.3. The ESI-MS, MS" and HILIC-MS analysis indicated that there was 10 glycans (H3N2,H3N2Xyl,H4N2,H3N2FucXyl,H4N2Xyl,H5N2,H6N2,H7N2,H8N2,H9N2) in both peanut total protein and allergen Ara h1. All of the glycans founded in Ara h1 are N-linked glycosylated. The glycan structures of Ara h1 can be divided into four types, in which 1 is core structure (3.58%),2 are complex type (22.07%),1 is hybrid type and 6 are high mannose type (45.25%). The mainly causative agents of allergen are those glycans which contains core ?1,3-Fuc structure with a content of 12.45% which fully proved that Ara h1 is the main allergenic protein, which contains the structure of carbohydrate determinants. The study aimed on structure analysis of glycans on allergenic protein Ara h1 provides an important information for elucidation of the functions and mechanisms of glycans in peanut allergy.