Collection of alpha1-acid glycoprotein molecular species by capillary electrophoresis and the analysis of their molecular masses and carbohydrate chains. Basic studies on the analysis of glycoprotein glycoforms.
A highly heterogeneous glycoprotein, alpha1-acid glycoprotein, was resolved into their glycoforms by capillary electrophoresis using a surface-modified capillary in 20 mM acetate buffer (pH 4.2) containing 0.5% (w/v) hydroxypropylmethylcellulose. We collected the fractions containing each glycoform as nearly pure state by capillary electrophoresis, and examined the molecular masses of these glycoforms by matrix assisted laser desorption time-of-flight mass spectrometry. We also analyzed carbohydrate chains after releasing them with N-glycosidase F followed by fluorescent labeling with 8-aminopyrene-1,3,6-trisulfonate. We found that the separation of glycoforms was mostly due to the presence of multiantennary carbohydrate chains. We propose that the present technique is useful for the analysis of post translational modification of proteins with carbohydrate chains.
Sei
Kazuo
K
Faculty of Pharmaceutical Sciences, Kinki University, Higashi-Osaka, Japan.
Nakano
Miyako
M
Kinoshita
Mitsuhiro
M
Masuko
Takashi
T
Kakehi
Kazuaki
K
D002240:Carbohydrate Sequence; D002241:Carbohydrates / Q000032:analysis* / Q000737:chemistry; D019075:Electrophoresis, Capillary / Q000379:methods*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009961:Orosomucoid / Q000737:chemistry / Q000302:isolation & purification*; D020033:Protein Isoforms / Q000737:chemistry / Q000302:isolation & purification*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D002241:Carbohydrates; D009961:Orosomucoid; D020033:Protein Isoforms