Frontal analysis capillary electrophoresis hyphenated to electrospray ionization mass spectrometry for the characterization of the antithrombin/heparin pentasaccharide complex.
The interaction of proteins with polysaccharides represents a major and challenging topic in glycobiology, since such complexes mediate fundamental biological mechanisms. A new strategy based on the hyphenation of frontal analysis capillary electrophoresis (FACE) with electrospray ionization mass spectrometry (ESIMS) is reported for the characterization of protein/carbohydrate complexes. While most of the previously reported CE-MS experiments were performed using capillary electrophoresis in zone format, we report for the first time CE-MS experiments in which CE was performed in frontal analysis (FACE-MS). We showed that the frontal mode offered a better sensitivity than zone mode and was well suited for the CE-MS coupling. This FACE-MS coupling was applied to the analysis of the complex between antithrombin and the sulfated pentasaccharide reproducing the antithrombin-binding sequence in heparin. The mixture of coincubated antithrombin and heparin pentasaccharide was continuously injected into the capillary, and the electrophoretic separation of the free and bound forms of the protein was achieved. The intact noncovalent complex antithrombin/heparin pentasaccharide was detected on-line by ESIMS in positive ionization mode and in nondenaturing sheath liquid conditions. The complex stoichiometry was determined from the mass measurement of the complex. In addition, the characterization of the sulfated pentasaccharide ligand dissociated from the complex was performed in negative ionization mode using a denaturing sheath liquid, allowing the determination of its molecular mass and sulfation features. This FACE-ESIMS strategy opens the way to ligand fishing experiments performed on heterogeneous carbohydrate mixtures and subsequent characterization of specifically bound carbohydrates.
Fermas
Soraya
S
CNRS UMR 8587, Université d'Evry-Val-d'Essonne, Laboratoire Analyse et Modélisation pour la Biologie et l'Environnement, F-91025 Evry, France.
Gonnet
Florence
F
Varenne
Anne
A
Gareil
Pierre
P
Daniel
Régis
R
D000991:Antithrombins / Q000032:analysis* / Q000737:chemistry; D001665:Binding Sites; D019075:Electrophoresis, Capillary / Q000379:methods*; D006493:Heparin / Q000031:analogs & derivatives / Q000032:analysis*; D008024:Ligands; D008970:Molecular Weight; D011134:Polysaccharides / Q000032:analysis* / Q000737:chemistry; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D013431:Sulfates / Q000737:chemistry
D000991:Antithrombins; D008024:Ligands; D011134:Polysaccharides; D013431:Sulfates; D006493:Heparin