Capillary electrophoresis-mass spectrometry for the identification of aminopyrene trisulfonic acid labeled glycans
The aim of this work was the development of capillary-electrophoresis/mass spectrometry (CE-MS) methods with high separation power for the characterization of aminopyrene-trisulfonic acid (APTS)-labeled glycans. State of the art for routine glycan analysis is capillary sieving electrophoresis with laser induced fluorescence detection (CSE-LIF) using the fluorescent APTS. However, a firsthand identification of glycans is not possible. This thesis presents two high performance CE-MS methods, with appropriate separation efficiency (i.e. resolution of structural isomers) for biopharmaceutical samples. The first method uses a novel alkaline background electrolyte (BGE) for the simultaneous analysis of APTS-labeled as well as charged native glycans. The alkaline BGE enables a robust CE-MS application and the determination of derivatization and ionization efficiency. The glycans of several different types of biopharmaceutically relevant proteins could be identified. The second CE-MS method, acidic BGE, was optimized in terms of separation efficiency and reproducibility by investigating potential system affecting parameters. Sheath liquid (SL) composition and pressure effects have the strongest impact on the desired high separation efficiency. Despite its sensitivity to pressure, the resulting method shows good reproducibility, since the main treats are clearly determined and pressure impacts can be counterbalanced. A systematic study on the signal assignment of glycans, analyzed by two different CSE-LIF methods and the proposed CE-MS methods, shows that the linear mobility correlation of glycans with similar functional groups within the two systems allows the signal assignment and identification of CE-LIF signals based on several known reference glycans. The development of a fritless, in-line solid phase extraction (SPE) setup, using a novel bead-string SPE design decreased the limit of detection of the acidic CE-MS method, from the sub-µM-range to a low nM-range.