Application of liquid chromatography/mass spectrometry and liquid chromatography with tandem mass spectrometry to the analysis of the site-specific carbohydrate heterogeneity in erythropoietin

被引:87
作者
Kawasaki, N [1 ]
Ohta, M [1 ]
Hyuga, S [1 ]
Hyuga, M [1 ]
Hayakawa, T [1 ]
机构
[1] Natl Inst Hlth Sci, Div Biol Chem & Biol, Setagaya Ku, Tokyo 1588501, Japan
关键词
ESI-LC/MS; ESI-LC/MS/MS; erythropoietin; carbohydrates;
D O I
10.1006/abio.2000.4739
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
High-performance liquid chromatography with electrospray ionization mass spectrometry (LC/MS) and liquid chromatography with tandem mass spectrometry (LC/MS/MS) were applied to the analysis of the site specific carbohydrate heterogeneity in erythropoietin (EPO) used as a model of the sialylated glycoprotein. N-linked oligosaccharides were released from recombinant human EPO expressed in Chinese hamster ovary cells enzymatically and reduced with NaBH4. Many different sialylated oligosaccharides of EPO were separated and characterized by LC/MS equipped with a graphitized carbon column (GCC). Glycosylation sites and the preliminary glycosylation pattern at each glycosylation site were determined by LC/MS of endoproteinase Glu-C-digested EPO. The detailed site-specific carbohydrate heterogeneity caused by the differences in the molecular weight, branch, linkage, and sequence was elucidated by GCC-LC/MS of the N-linked oligosaccharides released from the isolated glycopeptides. Structural details of the isomers were analyzed by LC/MS/MS, and it was indicated that di- and trisialylated tetraantennary oligosaccharides are attached to Asn24, 38, and 83, whereas their isomers, di- and trisialylated triantennary oligosaccharides containing N-acetyllactosamines, are combined with Asn24. Our method is useful for the determination of glycosylation sites, the site-specific carbohydrate heterogeneity of glycoproteins, and the carbohydrate structure. (C) 2000 Academic Press.
引用
收藏
页码:82 / 91
页数:10
相关论文
共 27 条
  • [1] ROLE OF GLYCOSYLATION ON THE SECRETION AND BIOLOGICAL-ACTIVITY OF ERYTHROPOIETIN
    DELORME, E
    LORENZINI, T
    GIFFIN, J
    MARTIN, F
    JACOBSEN, F
    BOONE, T
    ELLIOTT, S
    [J]. BIOCHEMISTRY, 1992, 31 (41) : 9871 - 9876
  • [2] A SYSTEMATIC NOMENCLATURE FOR CARBOHYDRATE FRAGMENTATIONS IN FAB-MS MS SPECTRA OF GLYCOCONJUGATES
    DOMON, B
    COSTELLO, CE
    [J]. GLYCOCONJUGATE JOURNAL, 1988, 5 (04) : 397 - 409
  • [3] HAYAKAWA T, 1991, DRUG BIOTECHNOLOGY R
  • [4] HIGUCHI M, 1992, J BIOL CHEM, V267, P7703
  • [5] PHYSICOCHEMICAL AND BIOLOGICAL CHARACTERIZATION OF ASIALOERYTHROPOIETIN - SUPPRESSIVE EFFECTS OF SIALIC-ACID IN THE EXPRESSION OF BIOLOGICAL-ACTIVITY OF HUMAN ERYTHROPOIETIN INVITRO
    IMAI, N
    HIGUCHI, M
    KAWAMURA, A
    TOMONOH, K
    OHEDA, M
    FUJIWARA, M
    SHIMONAKA, Y
    OCHI, N
    [J]. EUROPEAN JOURNAL OF BIOCHEMISTRY, 1990, 194 (02): : 457 - 462
  • [6] Analysis of carbohydrate heterogeneity in a glycoprotein using liquid chromatography mass spectrometry and liquid chromatography with tandem mass spectrometry
    Kawasaki, N
    Ohta, M
    Hyuga, S
    Hashimoto, O
    Hayakawa, T
    [J]. ANALYTICAL BIOCHEMISTRY, 1999, 269 (02) : 297 - 303
  • [7] KRANTZ SB, 1991, BLOOD, V77, P419
  • [8] APPLICATIONS OF ELECTROSPRAY MASS-SPECTROMETRY TO ERYTHROPOIETIN N-LINKED AND O-LINKED GLYCANS
    LINSLEY, KB
    CHAN, SY
    CHAN, S
    REINHOLD, BB
    LISI, PJ
    REINHOLD, VN
    [J]. ANALYTICAL BIOCHEMISTRY, 1994, 219 (02) : 207 - 217
  • [9] ROLE OF ANTENNARY STRUCTURE OF N-LINKED SUGAR CHAINS IN RENAL HANDLING OF RECOMBINANT-HUMAN-ERYTHROPOIETIN
    MISAIZU, T
    MATSUKI, S
    STRICKLAND, TW
    TAKEUCHI, M
    KOBATA, A
    TAKASAKI, S
    [J]. BLOOD, 1995, 86 (11) : 4097 - 4104
  • [10] Structural analysis of oligosaccharides derivatized with 4-aminobenzoic acid 2-(diethylamino)ethyl ester by matrix-assisted laser desorption/ionization mass spectrometry
    Mo, WJ
    Takao, T
    Sakamoto, H
    Shimonishi, Y
    [J]. ANALYTICAL CHEMISTRY, 1998, 70 (21) : 4520 - 4526