基于电喷雾电离质谱的人肝癌细胞HepG2与正常肝细胞L02的N-糖链的定性定量比较

doi:10.7503/cjcu20130960

摘要/Abstract

摘要：

以培养的原发性肝癌细胞HepG2和正常肝细胞L02为研究对象, 采用细胞裂解液提取总蛋白, 用PNGase F 酶解释放N-糖链, 以微晶纤维素柱结合石墨碳柱纯化分离N-糖链, 通过电喷雾电离质谱(ESI-MS)和串联质谱(MS/MS)对N-糖链进行序列鉴定, 以β-环糊精为内标对2种细胞系的N-糖链进行了定量比较分析. 结果表明, 在肝癌细胞系HepG2和正常细胞系L02中共检测到26种N-糖链, 与L02相比, HepG2的大多数高甘露糖型糖链、唾液酸化糖链和岩藻糖基化糖链的数量都明显升高, 其中有15种糖链在数量上具有极显著性差异(p<0.01), 1种糖链具有显著性差异(p<0.05). 本研究为进一步探索肝癌中各类N-糖链的表达特点及发现早期肝癌糖链标志物提供了参考.

关键词: 原发性肝癌细胞(HepG2), 正常肝细胞(L02), N-糖链, 电喷雾电离质谱

Abstract:

HepG2, a primary hepatocellular carcinoma cell line, and L02 derived from normal liver tissue, were chosen as model cell lines. In this work, the total proteins of HepG2 and L02 cells were extracted and enzymatically hydrolyzed by Peptide-N-glycosidase F(PNGase F). Then the released N-glycans were purified by microcrystalline cellulose and graphitized carbon columns. The structure of the purified N-glycans was identified by electrospray ionization mass spectrometry(ESI-MS)and MS/MS. To compare the difference of N-glycans between HepG2 and L02 cells, β-cyclodextrin was used as the internal standard to relative quantitative analysis of the N-glycans by MS. As results, 26 N-glycans are observed in both HepG2 and L02 cells. The amounts of high-mannose, sialylated, as well as fucosylated and sialylated N-glycans of HepG2 were higher than those of L02 cells. Among them, 15 kinds of N-glycans of HepG2 cells show very significant difference(p<0.01) and 1 kind of N-glycan show significant difference(p<0.05), compared to those of L02 cells. Our studies show potential in investigation of structure partterns of N-glycans expressed in hepatocellular carcinoma and in finding early biomarker in liver cancer diagnose.

Key words: Primary hepatocellular carionma cell line(HepG2 cell), Normal liver cell line(L02 cell), N-Glycans, Electrospray ionization mass spectrometry

中图分类号:

TrendMD:

潘丽英, 王承健, 袁江北, 张英, 黄琳娟, 王仲孚. 基于电喷雾电离质谱的人肝癌细胞HepG2与正常肝细胞L02的N-糖链的定性定量比较. 高等学校化学学报, 2014, 35(2): 237.

PAN Liying, WANG Chengjian, YUAN Jiangbei, ZHANG Ying, HUANG Linjuan, WANG Zhongfu. Qualitation and Quantitation for Comparative Analysis of N-glycans from Human Hepatocellular Carcinoma HepG2 and Normal Liver Cells L02 by Electrospray Ionization Mass Spectrometry^†. Chem. J. Chinese Universities, 2014, 35(2): 237.

图/表 4

参考文献 24

[1]	Apweiler R., Hermjakob H., Sharon N., Biochimica et Biophysica Acta(BBA)-General Subjects, 1999, 1473(1), 4—8
[2]	Furukawa K., Kobata A., Current Opinion in Biotechnology, 1992, 3(5), 554—559
[3]	Ohtsubo K., Marth J. D., Cell, 2006, 126(5), 855—867
[4]	Parodi A. J., Annual Review of Biochemistry, 2000, 69(1), 69—93
[5]	Helenius A., Aebi M., Science, 2001, 291(5512), 2364—2369
[6]	Isailovic D., Kurulugama R. T., Plasencia M. D., Stokes S. T., Kyselova Z. K., Goldman R., Mechref Y., Novotny M. V., Clemmer D. E., J. Proteome Res., 2008, 7(3), 1109—1117
[7]	Bai C., Wu G., Zhao L., Science China Chemistry, 2010, 53(4), 807—811
[8]	Skolnick A. A., The Journal of the American Medical Association, 1996, 276(18), 1458—1459
[9]	Marrero J. A., Clinics in Liver Disease, 2005, 9(2), 235—251
[10]	Gupta S., Bent S., Kohlwes J., Annals of Internal Medicine, 2003, 139(1), 46—50
[11]	Goldman R., Ressom H. W., Varghese R. S., Goldman L., Bascug G., Loffredo C. A., Abdel-Hamid M., Gouda I., Ezzat S., Kyselova Z., Mechref Y., Novotny M. V., Clinical Cancer Research, 2009, 15(5), 1808—1813
[12]	Xu N. S., Yang H. M., Cui M., Song F. R., Liu Z. Q., Liu S. Y., Chem. J. Chinese Universities, 2012, 33(11), 2430—2434
	(徐牛生, 杨洪梅, 崔勐, 宋凤瑞, 刘志强, 刘淑莹.高等学校化学学报,2012, 33(11), 2430—2434)
[13]	Orvisky E., Drake S. K., Martin B. M., Abdel-Hamid M., Ressom H. W., Varghese R. S., An Y., Saha D., Hortin G. L., Loffredo C. A., Goldman R., Proteomics, 2006, 6(9), 2895—2902
[14]	Ressom H. W., Varghese R. S., Goldman L., An Y., Loffredo C. A., Abdel-Hamid M., Kyselova Z., Mechref Y., Novotny M., Drake S. K., Goldman R., J. Proteome Res., 2008, 7(2), 603—610
[15]	Shimizu Y., Nakata., Kuroda Y., Tsutsumi F., Kojima N., Mizuochi T., Carbohydrate Research, 2001, 332, 381—388
[16]	Packer N. H., Lawson M. A., Jardine D. R., Redmond J. W., Glycoconjugate Journal, 1998, 15(8), 737—747
[17]	de Leoz M. L. A., An H. J., Kronewitter S., Kim J., Beecroft S., Vinall R., Lebrilla C., Disease Markers, 2008, 25(4), 243—258
[18]	Hua S., Williams C. C., Dimapasoc L. M., Ro G. S., Ozcan S., Miyamoto S., Lebrilla C. B., An H. J., Leiserowitz G. S., J. Chromatogr. A, 2013, 1279, 58—67
[29]	Kudo T., Nakagawa H., Takahashi M., Hamaguchi J., Kamiyama N., Yokoo H., Nakanishi K., Nakagawa T., Kamiyama T., Deguchi K., Nishimura S. I., Todo S., Molecular Cancer, 2007, 6(1), 32
[20]	Amano M., Yamaguchi M., Takegawa Y., Yamashita T., Terashima M., Furukawa J., Miura Y., Shinohara Y., Iwasaki N., Minami A., Nishimura S. I., Molecular & Cellular Proteomics, 2010, 9(3), 523—537
[21]	Wang C., Fan W., Zhang P., Wang Z., Huang L., Proteomics, 2011, 11(21), 4229—4242
[22]	Nakagawa T., Moriwaki K., Terao N., Nakagawa T., Miyamoto Y., Kamada Y., Miyoshi E., J. Proteome Res., 2012, 11(5), 2798—2806
[23]	Dennis J. W., Granovsky M., Warren C. E., Biochimica et Biophysica Acta(BBA)-General Subjects, 1999, 1473(1): 21—34
[24]	Taylor M.E., Drickamer K.; Translated by Ma Y. J., Introduction to Glycobiology, Chemical Industry Press, Beijing, 2006, 193—195
	(Taylor M.E., Drickamer K., 马毓甲[译]. 糖生物学导论, 北京: 化学工业出版社, 2006, 193—195)

Glycan type	m/z	Monosaccharide composition	Peak intensity ratio of N-glycan to β-cyclodextrin (mean±SD^a)		CV(%)		Relative ratio of N-glycans (HepG2 to L02)	Proposed structure
Glycan type	m/z	Monosaccharide composition	HepG2 (n=3)	L02 (n=3)			HepG2 (n=3)	L02 (n=3)
High-Mannose	933.25^b	H3N2	0.526±0.023	0.512±0.038	4.42	7.55	1.028
	1095.33^b	H4N2	0.673±0.059	0.680±0.036	8.81	5.33	0.990
	1257.33^b	H5N2	4.694±0.262	1.349±0.020	5.60	1.49	3.478
	1419.33^b	H6N2	4.011±0.241	3.755±0.233	6.01	6.21	1.068
	1581.33^b	H7N2	3.151±0.241	2.108±0.113	7.66	5.39	1.495
	1743.33^b	H8N2	3.547±0.278	3.127±0.136	7.86	4.37	1.134
	1905.50^b	H9N2	1.539±0.150	1.122±0.059	9.75	5.26	1.371
	964.17^c		1.403±0.053	1.037±0.119	3.79	11.47	1.352
Complex/hybrid	1110.25(-)^d	H5N4A2	0.611±0.070	0.333±0.013	11.51	4.11	1.834
(sialylated)	1293.25(-)^d	H6N5A2	2.597±0.102	1.108±0.181	3.94	16.38	2.344
	1315.75(-)^e		1.445±0.118	0.838±0.080	8.22	9.56	1.724
	1438.75(-)^d	H6N5A3	0.724±0.103	0.711±0.043	14.23	6.05	1.019
	1475.83(-)^d	H7N6A2	1.779±0.176	0.657±0.041	9.94	6.25	2.705
	1565.33(-)^f	H4N3A1	1.559±0.109	0.564±0.055	7.03	9.82	2.763
	1621.33(-)^d	H7N6A3	0.905±0.058	0.547±0.049	6.48	9.11	1.653
	1727.25(-)^f	H5N3A1	1.533±0.125	1.109±0.102	8.18	9.26	1.382
	1769.33(-)^f	H4N4A1	0.849±0.083	0.731±0.087	9.80	12.00	1.162
	1889.33(-)^f	H6N3A1	1.624±0.097	1.378±0.126	6.02	9.15	1.179
	1930.33(-)^f	H5N4A1	3.355±0.296	1.294±0.073	8.83	5.70	2.592
Complex/hybrid	1183.33(-)^d	H5N4A2F1	1.390±0.042	1.414±0.115	3.07	8.15	0.984
(fucosylated and	1366.33(-)^d	H6N5A2F1	6.244±0.478	3.798±0.036	7.66	0.95	1.644
sialylated)	1511.75(-)^d	H6N5A3F1	1.349±0.231	1.013±0.071	17.12	7.09	1.331
	1548.83(-)^d	H7N6A2F1	4.285±0.453	2.937±0.141	10.58	4.80	1.459
	1694.33(-)^d	H7N6A3F1	3.071±0.205	1.721±0.093	6.68	5.44	1.785
	1711.33(-)^f	H4N3A1F1	1.301±0.194	1.264±0.110	14.93	8.73	1.029
	1840.42(-)^d	H7N6A4F1	2.575±0.170	1.202±0.077	6.61	6.48	2.142
	1873.33(-)^f	H5N3A1F1	0.551±0.049	1.289±0.127	9.03	9.89	0.427
	1914.42(-)^f	H4N4A1F1	1.265±0.174	0.635±0.024	13.78	3.78	1.990

Glycan type	m/z	Monosaccharide composition	Peak intensity ratio of N-glycan to β-cyclodextrin (mean±SD^a)		CV(%)		Relative ratio of N-glycans (HepG2 to L02)	Proposed structure
Glycan type	m/z	Monosaccharide composition	HepG2 (n=3)	L02 (n=3)			HepG2 (n=3)	L02 (n=3)
High-Mannose	933.25^b	H3N2	0.526±0.023	0.512±0.038	4.42	7.55	1.028
	1095.33^b	H4N2	0.673±0.059	0.680±0.036	8.81	5.33	0.990
	1257.33^b	H5N2	4.694±0.262	1.349±0.020	5.60	1.49	3.478
	1419.33^b	H6N2	4.011±0.241	3.755±0.233	6.01	6.21	1.068
	1581.33^b	H7N2	3.151±0.241	2.108±0.113	7.66	5.39	1.495
	1743.33^b	H8N2	3.547±0.278	3.127±0.136	7.86	4.37	1.134
	1905.50^b	H9N2	1.539±0.150	1.122±0.059	9.75	5.26	1.371
	964.17^c		1.403±0.053	1.037±0.119	3.79	11.47	1.352
Complex/hybrid	1110.25(-)^d	H5N4A2	0.611±0.070	0.333±0.013	11.51	4.11	1.834
(sialylated)	1293.25(-)^d	H6N5A2	2.597±0.102	1.108±0.181	3.94	16.38	2.344
	1315.75(-)^e		1.445±0.118	0.838±0.080	8.22	9.56	1.724
	1438.75(-)^d	H6N5A3	0.724±0.103	0.711±0.043	14.23	6.05	1.019
	1475.83(-)^d	H7N6A2	1.779±0.176	0.657±0.041	9.94	6.25	2.705
	1565.33(-)^f	H4N3A1	1.559±0.109	0.564±0.055	7.03	9.82	2.763
	1621.33(-)^d	H7N6A3	0.905±0.058	0.547±0.049	6.48	9.11	1.653
	1727.25(-)^f	H5N3A1	1.533±0.125	1.109±0.102	8.18	9.26	1.382
	1769.33(-)^f	H4N4A1	0.849±0.083	0.731±0.087	9.80	12.00	1.162
	1889.33(-)^f	H6N3A1	1.624±0.097	1.378±0.126	6.02	9.15	1.179
	1930.33(-)^f	H5N4A1	3.355±0.296	1.294±0.073	8.83	5.70	2.592
Complex/hybrid	1183.33(-)^d	H5N4A2F1	1.390±0.042	1.414±0.115	3.07	8.15	0.984
(fucosylated and	1366.33(-)^d	H6N5A2F1	6.244±0.478	3.798±0.036	7.66	0.95	1.644
sialylated)	1511.75(-)^d	H6N5A3F1	1.349±0.231	1.013±0.071	17.12	7.09	1.331
	1548.83(-)^d	H7N6A2F1	4.285±0.453	2.937±0.141	10.58	4.80	1.459
	1694.33(-)^d	H7N6A3F1	3.071±0.205	1.721±0.093	6.68	5.44	1.785
	1711.33(-)^f	H4N3A1F1	1.301±0.194	1.264±0.110	14.93	8.73	1.029
	1840.42(-)^d	H7N6A4F1	2.575±0.170	1.202±0.077	6.61	6.48	2.142
	1873.33(-)^f	H5N3A1F1	0.551±0.049	1.289±0.127	9.03	9.89	0.427
	1914.42(-)^f	H4N4A1F1	1.265±0.174	0.635±0.024	13.78	3.78	1.990