Separation, identification, and interaction of heparin oligosaccharides with granulocyte-colony stimulating factor using capillary electrophoresis and mass spectrometry.
A capillary electrophoresis (CE) method was developed for the separation of heparin oligosaccharides compatible to study the interactions between the oligosaccharides and granulocyte-colony stimulating factor (G-CSF). Unfractionated heparin was eliminitively degraded to heparin oligosaccharides by an endolytic heparinase. The degraded smaller oligosaccharides (M(r) < 1000) were baseline-separated by CE under a 50 mM phosphate buffer (pH 9.0) in 10 min. Standard heparin disaccharides and larger oligosaccharides (1000 < M(r) < 8000) were all separated under optimized separation conditions. Compared with standard heparin disaccharides, smaller oligosaccharides contained one nonsulfated, two monosulfated, and two disulfated disaccharides, but trisulfated disaccharides were not found. The smaller oligosaccharides were also identified and molecular mass was deduced by electrospray ionization-mass spectrometry (ESI-MS). Furthermore, interactions between G-CSF and the oligosaccharides were studied by using capillary zone electrophoresis (CZE) under the above separation conditions. It was found that larger oligosaccharides could interact with G-CSF while smaller oligosaccharides were not observed to bind to G-CSF under the experimental conditions. In conclusion, the purified heparinase could selectively degrade heparin into oligosaccharides and the interaction between G-CSF and heparin was correlated with the chain length of heparin.
Liang
Aiye
A
Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, PR China.
Chao
Yapeng
Y
Liu
Xiaojun
X
Du
Yuguang
Y
Wang
Keyi
K
Qian
Shijun
S
Lin
Bingcheng
B
D002850:Chromatography, Gel / Q000379:methods; D004187:Disaccharides / Q000032:analysis; D019075:Electrophoresis, Capillary / Q000379:methods*; D016179:Granulocyte Colony-Stimulating Factor / Q000378:metabolism*; D006493:Heparin / Q000031:analogs & derivatives / Q000032:analysis / Q000302:isolation & purification* / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008353:Mannitol / Q000737:chemistry; D009844:Oligosaccharides / Q000032:analysis / Q000302:isolation & purification*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
D004187:Disaccharides; D009844:Oligosaccharides; C464536:heparin disaccharide; D016179:Granulocyte Colony-Stimulating Factor; D008353:Mannitol; D006493:Heparin