Studies On Amplification Of Mucin-Type O-glycans Based On Cellular Oligosaccharide Metabolism Engineering For Quali-Quantitative Analysis By Mass Spectrometry

Abnormal expressions of Mucin-type O-glycans are closely related to the occurrence and development of several diseases such as tumor.Thus,It is of great biological significance to systematically compare and analyze Mucin-type O-glycans in normal and tumor cells as to screen disease-associated Mucin-type O-glycans based on O-glycan microarray.However,Mucin-type O-glycans have a variety of core structures,which are complex and diverse.There is no template guidance for the synthesis of O-glycan with low expression level nature.Therefore,the methods to compare and analyze Mucin O-glycan structure in normal and tumor cells as well as to obtain Mucin O-glycan for the construction of glycan microarrays are still challenging.Therefore,based on the oligosaccharide metabolic engineering and isotope labeling techniques,combined with mass spectrometry and liquid chromatography-mass spectrometry(LC-MS)technology,we have developed these methods for which qualitative and quantitative analysis of Mucin-type O-glycans,identification of glycan isomers and amplification of O-glycans with active groups.The research results are as follows:Firstly,a relative quantitative analysis method for the stable isotope labeled Mucin-type O-glycans was established based on the oligosaccharide metabolism engineering technology.The established method demonstrated desirable feasibility,accuracy(relative error(RE)?4.20%),reproducibility(coefficient of variation(CV)?7.61%,n=3)and good quantitation linearity(R²?0.9984,n=3)for five Bn-O-glycans.The method has been successfully applied to quantitative analysis of the repertoire O-glycome changes between normal human liver cell line L02 and human hepatoma cell line SMMC-7721.The results indicated that SMMC-7721 cells express more glycans and higher levels of sialylated and fucosylated glycans over that of normal liver cells L02.Moreover,the?-2,3/2,6 sialic acid isomers of Bn-O-glycans from these two cells have been further quantitatively distinguished when involved a sialic acid specific derivatization procedure.Next,the Mucin-type O-glycans from L02 and SMMC-7721 were analyzed by online LC-MS/MS.The results showed that 14 O-glycans were detected in SMMC-7721 cells.Among them,three glycans have isomers,namely N1H1S1,N2H2S1 and N2H2F1S1 and N2H2 glycans not detected in the ESI-MS primary mass spectrometer were detected.13O-glycans were detected in L02 cells.Among them,four glycans isomers,which were assigned to be N1H1S1,N2H2,N2H2S1 and N2H2F1S1,were determined.Finally,a method based on cellular oligosaccharide metabolism engineering to amplify Mucin-type O-glycan carrying a functional group was developed.The results showed that 11Mucin-type O-glycans with alkyne group,which can be used in the construction of glycan microarrays,could be amplified and expressed by HeLa cells using peracetylated p-ethynylbenzyl-aminogalactose(?-ethynyl-Bn-O-Ac₃GalNAc)as a substrate.