Development Of Novel Methods To Analyze Glycosylation

Glycosylation is one of the most common forms of post translational modification(PTM)in eukaryotic proteins and involves the enzymatic attachment of glycans to asparagine(N-linked glycans)and serine or threonine(O-linked glycans).Glycosylation in proteins has been reported to have wide structural and functional roles such as protein folding,cel-cell recognition,cancer metastasis,and immune system activation et.al.The analysis o f N-glycosylation in proteins can be achieved with several levels of detail.The most simple and direct strategy is the release of the glycans from the protein followed by mass spectrometric(MS)analysis.Typically,N-glycans are released using enzymatic approaches.Peptide-N-glycosidase F(PNGase F),which releases unsubstituted and core 6-fucosylated N-glycans from proteins or peptides,has been used extensively in the N-glycomics studies of mammalians.Some further studies are performed with these released N-glycans directly without any modification.Or more usually,the released N-glycans are derived to enhance the separation and sensitivity.Here we have established a strategy to analyze N-glycans.We optimized deglycosylation conditions and reagents for fast release of the N-glycans,and labeled the glycans with a novel rapid labeling reagent called RapiFluor-MS,which can provide the benefits of effect separation,sensitive fluorescence and mass detection.Furthermore,,we established a hypothetical N-glycan database to simplify the data analysis.The complicated N-glycans analytical data can be assigned and compared automatically,accurately and efficiently.Finaly,we developed a novel strategy to analyze N –glycosylation with high sensitivity and efficiency.And has been successfully applied to analyze the N-glycans from different complex glycoproteins and cancer cel s.Besides,as there's no broad spectrum enzyme to release all kinds of O-glycans,we preliminary established a new method to analyze O-glycosylation.The enriched glycopeptides digested from glycoproteins were recognized with MS/MS in higher collision energy dissociation-product dependent-electron transfer dissociation(HCD-pd-ETD)mode.And the data was automaticly analyzed by Byonic software.With this method,we can get both O-glycosylation sites and the composition of the glycans.This method has been successfully applied to the O-glycosylation analysis of the recombinant human Integrin-binding bone sialoprotein(rhiBSP).And the information we got about the glycosylation sites and the composition of the glycans is more accurate and richer than using the traditional methods.