Establishment And Application Of Qualitative And Quantitative Analysis Of O-linked Glycans On Glycoprotein

Glycans exist on the surface of cells or biomacromolecules,which regulate the interaction between cells,cells and matrix,cells and molecules in the process of development of complex multicellular organisms,and even play significant roles among different organisms.In addition,there are also abundant glycans in the nucleus and cytoplasm.Thus,glycan must be considered as an important part of the cell biology study.Studies have found that a variety of tumor diagnostic markers and therapeutic targets are glycoproteins.Such studies could provide the evidence and theoretical basis for the diagnosis,treatment and prognosis of the tumor.O-linked glycans are a common protein glycosylation modification that often accompanies abnormal O-glycosylation during the development and progression of cancer.To study the aberrant O-glycosylation in tumorigenesis,tumor development and migration,the ultrafiltration membrane assisted enrichment and separation method(FASP)was established for dissociation and enrichment of total O-linked glycans on glycoproteins.A total of 90 O-linked glycans were identified by quantitative analysis in 5 bladder cell lines with different metastatic potentials.9,20,2,3,and 12 glycans were specifically found in HCV29,KK47,YTS-1,T24,and J82 cells,respectively.The O-linked glycans in A549 cells before and after hypoxia were also quantitatively analyzed,and 45 O-linked glycans were identified.39 and 29 specific O-linked glycans were identified in A549 cells before and after hypoxia.Among them,15 O-linked glycans were specifically found in normoxic cultured cells;5 glycans were specifically detected in hypoxic culture.23 glycans in normoxia and hypoxia cultured A549 cells were detected with different intensity.The levels of 11 O-linked glycans were decreased and which of 10 O-linked glycans were elevated in A549 cells after hypoxia.In order to more accurately study the differences in the expression of glycans.The O-linked glycans in lung cancer cells A549 cultured under normoxic and hypoxic were quantitatively analyzed using maltohexaose as internal glycan.A total of 44 O-linked glycans were identified by mass spectrometry analysis.Among them,21 glycans were detected both in cells under normoxia and hypoxic,and the intensity of maltohexaose granule was used for quantitatively analysis.It was found that the levels of 21 glycans were different in hypoxia.In summary,the significant changes occurred in identified glycans structure are mostly sialylated or fucosylated O-linked glycans,indicating the aberrant synthesis and degradation of sialylated and fucosylated glycan in the biological process and which is closely associated with the occurrence,development and deterioration of tumor.In this study,we hope to find the specific glycans in tumor cells,and provide preliminary basis for further study of the molecular mechanism between O-linked glycan and tumor development.