title: Partial filling affinity capillary electrophoresis using large-volume sample stacking with an electroosmotic flow pump for sensitive profiling of glycoprotein-derived oligosaccharides.
abstract: An online preconcentration technique, large-volume sample stacking with an electroosmotic flow pump (LVSEP) was combined with partial filling affinity capillary electrophoresis (PFACE) to realize highly sensitive analysis of the interaction of glycoprotein-derived oligosaccharides with some plant lectins. Oligosaccharides derivatized with 8-aminopyrene-1,3,6-trisulfonic acid (APTS) were delivered to an entire neutrally-coated capillary and then lectin solution was hydrodynamically introduced from the outlet of the capillary as a short plug. A negative voltage was then applied after immersion of both ends of the capillary in 100 mM Tris-acetate buffer, pH 7.0 containing 0.5% hydroxypropylcellulose as electrophoresis buffers. A low concentration of electrolytes in the sample solution causes a significant flow by electroendosmosis from anode to cathode and the APTS-labeled oligosaccharides move quickly towards the anode and concentrate in the lectin phase. Finally, electroosmotic flow becomes negligible when the capillary is filled with the background electrolyte delivered from the anodic reservoir and APTS-labeled saccharides pass through the lectin plug and are detected at the anodic end. If the APTS-labeled oligosaccharides are recognized by the lectin, the migration profiles should be altered. The sensitivity was enhanced by a factor of ca. 900 compared to typical hydrodynamic injection (3.45 kPa, 10s). By this method, increased residence time of APTS-saccharides in the lectin plug indicates highly efficient interaction with lectins, which differs completely from the results obtained by ordinary lectin PFACE. The run-to-run repeatability (n=18) of the migration time and peak area was high, with relative standard deviations of less than 0.7% and 6.1%, respectively.
authors.affiliation: Faculty of Pharmaceutical Sciences, Kinki University, 3-4-1 Kowakae, Higashi-osaka, Osaka, Japan. boo.0907@gmail.com
authors.forename: Eriko
authors.initials: E
authors.lastname: Fukushima
authors.forename: Yuki
authors.initials: Y
authors.lastname: Yagi
authors.forename: Sachio
authors.initials: S
authors.lastname: Yamamoto
authors.forename: Yumi
authors.initials: Y
authors.lastname: Nakatani
authors.forename: Kazuaki
authors.initials: K
authors.lastname: Kakehi
authors.forename: Takao
authors.initials: T
authors.lastname: Hayakawa
authors.forename: Shigeo
authors.initials: S
authors.lastname: Suzuki
mesh_terms: D000818:Animals; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D053841:Electroosmosis / Q000379:methods*; D019075:Electrophoresis, Capillary / Q000379:methods*; D006023:Glycoproteins / Q000737:chemistry*; D006801:Humans; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D037121:Plant Lectins / Q000378:metabolism; D011721:Pyrenes / Q000737:chemistry; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013552:Swine
chemical_list: C103430:8-aminopyrene-1,3,6-trisulfonic acid; D006023:Glycoproteins; D009844:Oligosaccharides; D037121:Plant Lectins; D011721:Pyrenes