title: Capillary electrophoresis/mass spectrometry of APTS-labeled glycans for the identification of unknown glycan species in capillary electrophoresis/laser-induced fluorescence systems.
abstract: The examination of protein glycosylation is of high importance, especially in the (bio)pharmaceutical sector. The analysis of protein glycosylation is conducted routinely in high performance by capillary electrophoresis with laser-induced fluorescence (CE/LIF) using 8-aminopyrene-1,3,6-trisulfonic acid (APTS)-labeled glycans. In this work we present an optimized capillary electrophoresis/time-of-flight mass spectrometry (CE/TOF-MS) methodology for these labeled glycans, which combines the high separation performance of CE with the high resolution, accuracy, and speed of TOF-MS for eased glycan identification. The system based on an acidic background electrolyte (BGE) provides a migration direction analogue to routine CE/LIF systems. Different BGE compositions, capillary dimensions, coatings, and instrumental parameters were tested to optimize the system with respect to separation efficiency and robustness. Subsequently, the CE/MS method optimized for acidic conditions was compared to an alkaline CE/MS method. Further, the mobilities of six APTS-labeled complex-type N-glycans were compared for both CE/MS methods and two standard CE/LIF approaches. For the acidic and alkaline BGE systems, the mobilities of sialylated glycans were shifted relative to nonsialylated glycans in comparison to common CE/LIF systems. However, in this study a straightforward unequivocal peak assignment was achieved for all unknown glycans in a medium complex glycan mixture from a fusion protein.
authors.affiliation: Aalen University , Aalen, 73430 Germany.
authors.forename: Svenja-Catharina
authors.initials: SC
authors.lastname: Bunz
authors.forename: Erdmann
authors.initials: E
authors.lastname: Rapp
authors.forename: Christian
authors.initials: C
authors.lastname: Neusüss
mesh_terms: D019075:Electrophoresis, Capillary / Q000379:methods*; D013058:Mass Spectrometry / Q000379:methods*; D011134:Polysaccharides / Q000737:chemistry*; D011721:Pyrenes / Q000737:chemistry*; D013050:Spectrometry, Fluorescence / Q000379:methods*
chemical_list: C098210:1-aminopyrene-3,6,8-trisulfonic acid; D011134:Polysaccharides; D011721:Pyrenes