Characterization of glycopeptides from recombinant coagulation factor VIIa by high-performance liquid chromatography and capillary zone electrophoresis using ultraviolet and pulsed electrochemical detection.
Four glycopeptide (GP) fractions containing glycosylated Asn 322 were isolated from a tryptic digest of recombinant coagulation factor VII by reversed-phase-HPLC (RP-HPLC). Characterization of the GPs by enzymatic desialylation and RP-HPLC as well as by enzymatic deglycosylation, RP-HPLC, and high-pH anion-exchange chromatography indicated that the four GPs consisted of the same decapeptide but with 0, 1, 2, or 4 residues of sialic acid. In comparison to HPLC, capillary zone electrophoresis (CZE) using uv and pulsed electrochemical detection (PED) afforded improved separation of GPs from each other and from contaminants. CZE-uv and CZE-PED of the desialylated GPs and deglycosylated GPs corroborated the results obtained with the chromatographic methods.
Weber
P L
PL
Center for Bioanalytical Research, University of Kansas, Lawrence 66047, USA.
Kornfelt
T
T
Klausen
N K
NK
Lunte
S M
SM
D000595:Amino Acid Sequence; D001216:Asparagine; D002197:Capillary Action; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D002852:Chromatography, Ion Exchange / Q000379:methods; D004563:Electrochemistry / Q000379:methods; D004586:Electrophoresis / Q000379:methods; D015942:Factor VIIa / Q000737:chemistry*; D006020:Glycopeptides / Q000737:chemistry* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry; D014357:Trypsin
D006020:Glycopeptides; D011994:Recombinant Proteins; D001216:Asparagine; D015942:Factor VIIa; D014357:Trypsin