The Separation And Purification Of Lycium Glycoprotein LbGp5-B And N-glycans Structure Analysis

Glycoprotein LbGp5-B is a kind of Glycoconjugates, it is isolated from the Lycium barbarum L polysaccharides (LBP), has antioxidant activity in vitro, accounting for 0.019% of the Lycium barbarum L. However, for now, we never found the report that the analysis of glycan structure on protein.In this paper, we extracted LBP from the Lycium barbarum L at first, then have get the homogeneous of glycoprotein LbGp5-B how isolated and purified many times of LBP. Using SDS-PAGE tested the purity and molecular weight of protein, using the Phenol-sulfuric acid method (Phenol-sulfuric acid) and coomassie brilliant blue method (Bradford) and gas chromatography (GC) method for determining the basic physical and chemical properties. Using PNGaseA and PNGaseF released N-glycans on the glycoprotein, using ESI-MS and MSn analysis the structure of the glycoprotein. The conclusion is as follows:1. The identification of SDS-PAGE show the glycoprotein LbGp5-B is homogeneous and the molecular weight is about 21KDa.2. The Phenol-sulfuric acid and Bradford analysis show:glycoprotein LbGp5-B sugar content of 9.6%, the protein content of 56.13%.32.47% of the remaining is pigments.3. Using ESI-MS, MS/MS and MS" analysis show:In total,18 N-glycans corresponding and 19 N-glycan compositions were found in glycoprotein LbGp5-B, of which 1 is core structure (5.26%),2 are non-core structure (10.53%),3 are hybrid type (15.79%),6 are high mannose type (31.58%),7 are complex type (36.84%). In the complex type have one is single antenna (14.92%),3 are double antenna (42.86%), one is tridipole antenna (14.92%). The composition of H3N4 has two isomers. All of the N-glycans are neutral in the protein. It has two N-glycans had been fucosylated and never find the N-glycans of Sialyl. In this paper, we can offer some basics for the relation between the protein function and glycosilation by test and analysis the glycoprotein LbGp5-B.