Qualitation and Quantitation for Comparative Analysis of N-glycans from Human Hepatocellular Carcinoma HepG2 and Normal Liver Cells L02 by Electrospray Ionization Mass Spectrometry†

doi:10.7503/cjcu20130960

Abstract

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

CLC Number:

TrendMD:

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^†[J]. Chem. J. Chinese Universities, 2014, 35(2): 237.

Figures/Tables 4

References 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