| Glycosylation, one of the most common post-translational protein modifications, acts an important role in eukaryotic systems. With the continuous development of biological mass spectrometry technology, profiling the specific glycan as the tumor biomarkers has attracted the interest in glycobiology research.In this study, we chose SMMC-7721, a human hepatocellular carcinoma cell line, and L02 from human normal liver tissue as our objects materials. The total proteins of SMMC-7721 and L02 were extracted through the conjunction of RIPA Lysis buffer and mechanical disruption. The N-glycans were released from SMMC-7721 and L02 total protein by enzymatically hydrolysis. We used ultra-filtration to separate the released N-glycan and protein with O-glycan. The O-glycan from SMMC-7721 and L02 cells were released by "one pot". And all the N-glycan and O-glycan has been derived and purified to identify their structures and quantify by ESI-MS, MS/MS and HILIC-MS. Our study got comparisons between the quality and quantity data of N-glycan and O-glycan from SMMC-7721 and L02 total protein, which will be reference for further explore the character of glycan liver cancer for biomarkers. The major research results were concluded as follows:Comparison analysis of N-glycans from SMMC-7721 and L02 total protein revealed that: (1)14 N-glycans were detected in both SMMC-7721 and L02, of which 7 were oligomannose type,4 were complex type and 3 were hybrid type. (2) And the amount of oligomannose N-glycans of SMMC-7721 were lower than those of L02. T test results showed that 4 oligomannose existed very significant difference(p<0.01). However, the amount of complex and hybrid N-glycans of SMMC-7721 were higher than those of L02, and 9 N-glycan showed very significant difference(p<0.01) through T text results.1 N-glycan showed significant difference(p<0.05). (3) In addition,8 N-glycan (H5N5, H6N5, H7N6, H3N3F1, H5N4F1, two isomers of H6N5F1, H7N6F1) were observed only in SMMC-7721. Their terminal were all modified by Gal-GlcNAc (expect H3N3F1 which is in low level). Five of them occurred core fucosylation and H5N5 belongs to bisecting N-acelyglucosamine. (4) On the other side, we found that in SMMC-7721, the greatest number and the greatest amount of N-glycan type variety is complex. But in L02 cells, whether the number of N-glycan type variety or the amount of N-glycan type was oligomannose. (5) Besides, we found multiple antenna structure in SMMC-7721 complex type, including triple and tetra antenna, and no similar structure in L02.Comparison analysis of O-glycans revealed that:(1)4 O-glycans were detected in both SMMC-7721 and L02, of which 2 were non-sialylated type,1 were only sialylated type and 1 were sialylated and fucosylated type. (2) And the amounts of 7 O-glycans from SMMC-7721 were higher than those of L02. T test results showed that,5 O-glycans showed very significant difference (p<0.01), and 1 only sialylated O-glycan showed significant difference (p<0.05). (3) However, the amount of H1N1 of SMMC-7721 were lower than L02, and its T text result showed very significant difference(p<0.01). In addition,4 O-glycan (H1N1A1, H1N1A2, H2N2A1, H2N2A2) were observed only in SMMC-7721. In particular, H1NN1A1 and H1N1A2 were belong to sialyl T-antigen and diSialyl T-antigen, which have been certified as tumor associated antigen. And the other two O-glycans also occurred terminal sialyaltion. (4) In addition, we found that in SMMC-7721, whether the number of O-glycan type variety or the amount of O-glycan type were only sialylated type. However, no matter the number of O-glycan type variety or the amount of O-glycan type were non-sialylated neutrual type in L02. |
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