Capillary zone electrophoretic resolution of recombinant human bone morphogenetic protein 2 glycoforms. An investigation into the separation mechanisms for an exquisite separation.
Recombinant human bone morphogenetic protein 2 (rhBMP-2) is a disulfide-linked homodimeric glycoprotein (M(r) = 30,000) which induces bone formation in vivo in several animal model systems. In this paper, we report the separation of a homogeneous rhBMP-2 sample into nine peaks by capillary zone electrophoresis (CZE), using a simple, pH 2.5, phosphate buffer containing no additives. The nine peaks have been identified to be glycoforms of rhBMP-2 [designated as (rhBMP-2)2-(GlcNAc)4(ManZ), where Z varies from 10 to 18]. The difference between any adjacent pair of peaks is only one mannose residue (M(r) = 162). The ability of CZE to resolve rhBMP-2 glycoforms having the same charge and differing only 0.5% in molecular mass, without resorting to chemical complexation, is both unexpected and intriguing. Possible mechanisms explaining how the additional mannose can affect the mobility of rhBMP-2 glycoforms were explored. Zeta potentials of various glycoforms were calculated from their mobilities and interpreted in light of diffuse double layer parameters. Our results suggest that CZE employing a low-pH buffer, where proteins are highly charged, may be uniquely suitable for complex protein glycoform analysis.
Yim
K
K
Genetics Institute, Andover, MA 01810, USA.
Abrams
J
J
Hsu
A
A
D000595:Amino Acid Sequence; D019485:Bone Morphogenetic Proteins; D018360:Crystallography, X-Ray; D019075:Electrophoresis, Capillary; D006801:Humans; D006868:Hydrolysis; D007202:Indicators and Reagents; D007536:Isomerism; D008358:Mannose / Q000737:chemistry; D008745:Methylation; D008956:Models, Chemical; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D011134:Polysaccharides / Q000032:analysis; D011506:Proteins / Q000737:chemistry* / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D016339:Spectrometry, Mass, Fast Atom Bombardment; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D019485:Bone Morphogenetic Proteins; D007202:Indicators and Reagents; D011134:Polysaccharides; D011506:Proteins; D011994:Recombinant Proteins; D008358:Mannose