Preparation of a Concanavalin A Immobilized Affinity Column and Its Application in the Structural Analysis of Ribonuclease B
伴刀豆球蛋白亲和色谱柱的制备及其在糖蛋白核糖核酸酶B结构分析中的应用

The research on glycoproteomes represents an interesting field in the functional proteomics research. Affinity chromatography and mass spectrometry are powerful techniques that are used for gaining valuable information on glycoproteomes because glycoproteins and their unusual forms resulting from protein glycosylation can be important indicators of several diseases. In this study, the concanavalin A (Con A) immobilized silica packing was prepared and used for the separation of glycoprotein and glycopeptides. A very low, non-specific adsorption on the Con A affinity column was demonstrated by mass recovery of bovine serum albumin at more than 98.5%. The effect of concentration of methyl-alpha-D-mannopyranoside (alpha-Me-D-Man) in the mobile phase and the effect of flow rate on the retention behavior of ribonuclease B (RNase B) were also investigated. The standard glycoprotein RNase B was separated under optimized conditions using 0.2 mol/L alpha-Me-D-Man in the mobile phase at a flow rate of 0.5 mL/min. Meanwhile, the oligosaccharides and glycopeptides were enriched using a Con A column after digestion of the purified RNase B with peptide-N-glycosidase F (PNGase F) and trypsin. The structure of N-linked glycan and the rate and the site of glycosylation of RNase B were determined by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS). Glycoproteins and glycopeptides in human serum and digest solution could be separated by this method. The results showed that this method is rapid and sensitive for the purification and characterization of glycoproteins and glycopeptides.