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Abstract:
The analysis of protein glycosylation became of increasing importance in glycobiological research, as well as for bioprocess monitoring during development and production of biopharmaceuticals, in the recent past. Hence, the systematic study and characterization of the broad variety of glyco-moieties in different cell types and organisms demands for sophisticated analytical tools and dedicated databases in glycobiology, respectively glycobiotechnology. In order to enhance and improve the comparatively small existing glycoanalytical toolbox, automated high-throughput (HT) and high-resolution analysis methods with automated data processing and analysis are required. Besides several mass spectrometry and liquid chromatography based analysis techniques, electrokinetic separation techniques for the analysis of oligosaccharides became apparent during the recent past. Especially, multiplexed capillary gel electrophoresis with laser induced fluorescence detection (xCGE-LIF), a quite feasible electromigrative separation technique - using standard DNA sequencer equipment - has been developed for HT- glycoprofiling of APTS-labeled glycans [1,2]. The application of this technique with up to 96 capillaries in parallel, results in massive reduction of the effective separation time per sample combined with an impressive sensitivity achieved due to LIF detection [3]. Due to the speed of the xCGE-LIF based method itself, the analysis of the enormous amount of upcoming data became the HT-bottleneck. Until now, no bioinformatics software tools have been available to automatically process and analyze the xCGE-LIF generated glyco-data. To tackle this issue, the automated analysis software “glyXtool” for xCGE-LIF based data was developed. The software comes as a platform-independent distribution with a user-friendly graphical interface, that supports data-processing, -analysis and -interpretation. The software provides automated raw data smoothing, baseline correction and the normalization to an internal standard, enabling structural elucidation and annotation by referring to a corresponding oligosaccharide-database. Furthermore, a peak comparison function, as well as several implemented statistical tools facilitate HT-screening for biomarkers in large sample sets. The resulting high-performance system (method, software & database) enables fully automated, highly sensitive, instrument-, lab- and operator-independent “real” HT-glycoanalysis in high-resolution, even when operated by non-experts.
