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Application of capillary electrophoresis, liquid chromatography, electrospray‐mass spectrometry and matrix‐assisted laser desorption/ionization ‐ time of flight ‐ mass spectrometry to the characterization of recombinant human erythropoietin
Author(s) -
Zhou GuoHua,
Luo GuoAn,
Huang LeQun,
Zhou Yong,
Zhang XiaoDano,
Zhou KeYu
Publication year - 1998
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.1150191315
Subject(s) - chromatography , chemistry , mass spectrometry , capillary electrophoresis–mass spectrometry , capillary electrophoresis , electrospray ionization , sample preparation in mass spectrometry , protein mass spectrometry , electrospray , time of flight mass spectrometry , high performance liquid chromatography , matrix assisted laser desorption/ionization , sialic acid , desorption , ionization , biochemistry , ion , organic chemistry , adsorption
Abstract High performance capillary electrophoresis (HPCE), high performance liquid chromatography (HPLC), matrix‐assisted laser desorption/ionization ‐ time of flight ‐ mass spectrometry (MALDI‐TOF‐MS), on‐line CE‐electrospray ionization‐mass spectrometry (CE‐ESI‐MS) and on‐line LC‐ESI‐MS have been employed to characterize a heterogeneous glycoprotein, recombinant human erythropoietin (rHuEPO) expressed from Chinese hamster ovary (CHO) cells. The analysis was demonstrated through two specific levels of detail: the intact protein and tryptic digests of the protein. Six glycoforms of rHuEPO were separated by HPCE; seventeen tryptic fragments in a total of 21 nonglycosylated and glycosylated peptides were characterized; the O ‐linked glycopeptides were analyzed directly by CE‐ESI‐MS and LC‐ESI‐MS. In particular, four glycans of O ‐acetylation of sialic acid were identified in the O ‐linked glycosylated fragments. The molecular weight of rHuEPO was accurately determined by MALDI‐TOF‐MS.