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.