Qualitation And Quantitation For Comparative Analysis Of N-/O-glycans From Human Hepatocellular Carcinoma HepG2and Normal Liver Cells L02by Electrospray Ionization Mass Spectrometry

Glycosylation is one of the most important post-translational modifications of protein, it is estimated that over50%of human proteins are glycosylated. The important role of abnormal glycosylation is more and more recognized in cancer biology, Using glycomics analytical method to identify glycan biomarkers of cancers has stimulated growing interests into glycobiology research in recent years. With its rapid development, biological mass spectrometry technology has been widely used in the study of glycomics and proteomics, therefore, it provides possible to screen highly specific glycan biomarkers. In this work, HepG2, a human primary hepatocellular carcinoma cell line, and L02derived from liver tissue, were chosen as model cell lines. The total proteins of HepG2and L02cells were extracted, the N-glycans were released from HepG2and L02by enzymatically hydrolysis and the O-glycans were released from HepG2and L02by Carlson reductive β-elimination, respectively. The structure of the purified N-/O-glycans was identified by electrospray ionization mass spectrometry (ESI-MS) and tandem mass spectrometry (MS/MS), then qualitation and quantitation for comparative analysis of N-/O-glycans from HepG2and L02by internal standard mass spectrum quantitative method. Our studies show potential in investigation of structure partterns of N-/O-glycans expressed in human hepatocellular carcinoma and in finding early biomarker in liver cancer diagnose. Research results obtained are summarized as following:1. N-glycans were purified and separated by graphitized carbon column, the separation method of neutral and acidic glycans was established by optimizing the conditions, then, the method can well avoid the ionization suppression phenomena of the high abundance glycans to the low abundance glycans by mass spectrometry detection.2. The analysis results of N-glycans showed that26N-glycans were observed in both HepG2and L02cells, of which7glycans were high-mannose,10glycans were sialylated glycans and9were fucosylated and sialylated N-glycans, In addition, the amounts of N-glycans of HepG2were higher than those of L02cells. T test results show that, Among them,15N-glycans of HepG2cells show very significant difference(p﹤0.01) and1N-glycan shows significant difference(p﹤0.05), compared to those of L02cells.3. The analysis results of O-glycans revealed that10O-glycans were observed in HepG2cell line and9O-glycans were detected in L02cell line. In this study, we observed1truncated O-glycan was assigned as H1A1(NeuAc-GalNAc, sialyl Tn antigen) in HepG2cells, Several mechanisms have been proposed that lead to a truncation of O-glycan is ubiquitous in cancer cells, in addition, of which9O-glycans are observed in both HepG2and L02cells. T test results show that, Among them,5O-glycans of10O-glycans in HepG2cells show very significant difference(p﹤0.01) and2O-glycans show significant difference (p﹤0.05), compared to those of L02cells.