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Towards Complex Characterization of Glycomic Samples Using Combination of HILIC with CZE in Microfluidic System
Authors: Česla Petr | Vaňková Nikola | Váňová Jana | Nováková Zuzana | Fischer Jan
Year: 2015
Type of publication: ostatní - článek ve sborníku
Name of source: Applied Natural Sciences 2015: conference proceedings
Publisher name: Univerzita sv. Cyrila a Metoda v Trnave
Place: Trnava
Page from-to: nestránkováno
Titles:
Language Name Abstract Keywords
eng Towards Complex Characterization of Glycomic Samples Using Combination of HILIC with CZE in Microfluidic System Glycosylation is important posttranslation modification of proteins, which is correlated to various pathological states. The analysis of glycosylation patterns can be challenging task and may require implementation of powerful separation techniques coupled to mass spectrometry for unequivocal identification of glycan structures. In present work, we have focused on development of separation methods suitable for characterization of complex glycan mixtures. Thus we have optimized hydrophilic interaction chromatographic separation of glycans and maltooligosaccharides as standards for calibration of retention scale on silica gel, amino-, amide- and zwitterionic stationary phases. The glycans and oligosaccharides were fluorescently labelled by reductive amination with 8-aminonaphthalene-1,3,6-trisulfonic acid, 2-aminobenzoic acid and 2-aminobenzamide. The contributions of structural units and the influences of mobile phase additives (acetic acid, ammonium acetate) to selectivity and retention have been described using several models. The capillary zone electrophoretic separation in connection with mass spectrometry was optimized using volatile separation buffers and sheath-flow interface. Capillary separations were compared to the separations in borosilicate-glass microchip platform. Both methods were coupled in two-dimensional system via microfluidic interface capable of transferring the selected fractions between separation systems yielding significantly increased peak capacity. The laser induced fluorescence detection has been utilized by confocal detector build in laboratory using 3D printing technology. The applicability of the separation system was demonstrated on analyses of deglycosylation products of hIgG and fetuin. hydrophilic interaction liquid chromatography; capillary zone electrophoresis; glycans; oligosaccharides; fluorescence detection; microfluidic separations

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