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

• Published in: Electrophoresis Vol. 19; no. 13; p. 2348
• Main Authors: Zhou, G H, Luo, G A, Zhou, Y, Zhou, K Y, Zhang, X D, Huang, L Q
• Format: Journal Article
• Language: English
• Published: Germany 01.10.1998
• Subjects: Amino Acid Sequence; Animals; CHO Cells; Chromatography, High Pressure Liquid; Cricetinae; Electrophoresis, Capillary; Erythropoietin - analysis; Erythropoietin - chemistry; Erythropoietin - metabolism; Glycosylation; Humans; Mass Spectrometry - methods; Molecular Weight; Peptide Fragments - analysis; Peptide Fragments - chemistry; Recombinant Proteins; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization - methods; Trypsin - metabolism
• ISSN: 0173-0835
• DOI: 10.1002/elps.1150191315

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.