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.
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.
Zhou
G H
GH
Department of Chemistry, Tsinghua University, Beijing, PRC.
Luo
G A
GA
Zhou
Y
Y
Zhou
K Y
KY
Zhang
X D
XD
Huang
L Q
LQ
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D002851:Chromatography, High Pressure Liquid; D006224:Cricetinae; D019075:Electrophoresis, Capillary; D004921:Erythropoietin / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods; D008970:Molecular Weight; D010446:Peptide Fragments / Q000032:analysis / Q000737:chemistry; D011994:Recombinant Proteins; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D014357:Trypsin / Q000378:metabolism
D010446:Peptide Fragments; D011994:Recombinant Proteins; D004921:Erythropoietin; D014357:Trypsin