Magnetic Particle Enrichment Of Glycoproteins / Glycopeptides New Method

Protein glycosylation and glycoproteomics are growing fields of interest due to the relationship between glycosylation and the structure and function of glycoproteins as well as that between glycosylation degree and the health status of cells. The enrichment of glycopeptides/glycoproteins and identification of glycosylation sites/glycoproteins are important contents in glycoproteomics. Many technologies of proteomics have been applied to the analysis of glycosylation primarily, but the characteristic of the glycosylation determines the high requirement to the analysis technologies. Since the technologise now existed is still insufficient, new technologies for glycoproteins analysis are stringent necessity.Here we developed two new methods for the enrichment of glycoproteins/ glycopeptides. Both methods combined the magnetic microspheres technology with hydrazide chemistry and Lectin, respectively. The hydrazide-functionized magnetic microspheres and GNA-functionized magnetic microspheres are prepared correspondingly. The condition for capturing glycopeptides by hydrazide-functionized magnetic microspheres and immobiling GNA onto the magnetic microspheres was evaluated and optimized, and the specificity and validity of two methods was demonstrated by using standard glycoproteins sample. The experimental results showed that the specificity and validity of two methods was all right. A new analysis system for enrichment of glycoproteins/glycopeptides as well as the identification of glycoproteins/glycosylation sites was also established by coupling enrichment of glycopeptides by hydrazide- functionized magnetic microspheres with release of polysaccharide chains by PNGase F and mass spectrometry as well.Experimental results showed that the glycoproteins/glycopeptides enrichment method developed here is simple and repid. Since the nonglycoproteins and non-high-mannose type glycoproteins were removed in the process of both glycoproteins enrichment by GNA-functionized magnetic microspheres, and the nonglycopeptides was also moved away by hydarzide chemistry method, the complexity of the samplewas reduced hugely by both methods, which would reduce the difficulty of posteriordata processing. So it may achieve the high and automatization of identification ofglycoproteins. The study layed a foundation for both methods to be used in theglycosylation analysis of complex samples.