Quali-quantitative Profiling of Mucin-type O-Glycans in Normal and Tumor Cell Lines Based on Oligosaccharide Metabolic Engineering Combined with Mass Spectrometry†

doi:10.7503/cjcu20180143

Abstract

Abstract:

The O-glycans on glycoproteins have important biological functions, and their abnormal expressions are closely related to the occurrence and development of several diseases such as tumor. Thus, it is of importance to conduct quali-quantitative profiling of O-glycans expression in normal and disease cells. However, due to the structural complexities and inhomogeneous expression of O-glycans, quali-quantitative analyses of these glycoconjugates still encounter challenges. In this study, based on the oligosaccharide metabolic engineering combined with mass spectrometry, we have profiled and analyzed O-glycans expressed in four tumor cell lines(HeLa, SMMC-7721, HepG2 and MCF-7) and one normal cell lines(L02) quali-quantitatively. The results showed that 19, 11, 6 and 5 O-glycans were observed in four cancer cell lines, HeLa, SMMC-7721, HepG2 and MCF-7 respectively. And 10 O-glycans were detected in one normal cell line(L02). Qualitative and relative quantitative comparison of O-glycans expressed in tumor and normal cell lines showed that three O-glycans(N1, H1N1A1 and H1N1A2) were observed in all cell lines. The number of O-glycans expressed in cancer cell lines is more than those in normal cells. Moreover, these O-glycans were highly fucosylated and sialylated. Further MS/MS data showed that these O-glycans were belonged to Core2 type Mucin O-glycans and their sugar compositions were H3N3F1A2, H4N4F1A2 and H5N5F1A1(fucosylated O-glycans) and H5N4A1, H4N4A2 and H5N5A2(sialylated O-glycans).

Key words: Mucin-type O-glycan, Tumor cell, Normal cell, Oligosaccharides metabolic engineering, Mass Spectrometry

CLC Number:

O657

TrendMD:

YANG Mingming,NAN Lijing,JIN Wanjun,WANG Chengjian,HUANG Linjuan,ZHANG Ying,WANG Zhongfu. Quali-quantitative Profiling of Mucin-type O-Glycans in Normal and Tumor Cell Lines Based on Oligosaccharide Metabolic Engineering Combined with Mass Spectrometry^†[J]. Chem. J. Chinese Universities, 2018, 39(9): 1904.

Figures/Tables 6

Fig.1 Flow diagram of quali-quantitative analysis of mucin type O-glycans in normal and tumor cells based on oligosaccharide metabolic engineering techniques(A) The per-acetylation of GalNAc-α-O-Bn; (B) amplification of O-glycans through cell oligosaccharide metabolic engineering and subsequent MS profiling

Fig.2 ESI-MS spectra of O-glycans generated through reducing β-elimination(A) and cell oligosaccharide metabolic engineering(B) and MS/MS spectrum of Bn-O-glycans with m/z 1200.33(C) in SMMC-7721 cells* Impurities. Red characterized numbers represent the target O-glycan signals in [M+Na]+ ioned peaks; blue characterized numbers mean the free oligosaccharide impurities.

Fig.3 MALDI-TOF-MS analysis of Bn-O-glycan secreted in HeLa(A), SMMC-7721(B), HepG2(C) and MCF-7(D) cellsBlack characterized numbers mean the target O-glycan signals in [M+Na]+ ioned peaks; blue characterized numbers present the target O-glycan signals in [M+K]+ ionized peaks.

Fig.4 MALDI-TOF-MS analysis of Bn-O-glycan secreted in L02 cell line* Impurities. Black characterized numbers represent the target O-glycan signals in [M+Na]+ ionized peaks.

Fig.5 Relative quantification and comparison of Bn-O-glycans in two tumor liver cells(SMMC-7721, HepG2) and one normal liver cells(L02)

Fig.6 Relative quantification and comparison of O-glycans expressed in four tumor cells HeLa, SMMC-7721, HepG2 and MCF-7 and one normal cell(L02)

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