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Zusammenfassung:
Glycomics is a rapidly emerging field that can be viewed as a continuation of the genomic and proteomic era. Hence, there is a dramatic dynamic increase in the demand for sophisticated databases and analytical tools in glycobiology respectively glycobiotechnology. In order to enhance and improve the comparatively small existing glycoanalytical toolbox, a fully automated, high-throughput (HTP) and high-resolution (HR) analysis method, with a fully automated data evaluation, is required. Besides several mass spectrometry and liquid chromatography based analysis techniques, electromigrative separation techniques for the analysis of oligosaccharides became apparent during the recent past. Especially capillary gel electrophoresis with laser induced fluorescence detection (CGE-LIF) as one feasible electromigrative separation technique - using standard DNA sequencer equipment - has been developed for HTP 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 lack of appropriated software, the Matlab® based software tool “glyXtool V3.0” was developed for the data analysis of CGE-LIF generated electropherogram data. Thereby, glyXtool V3.0 provides automated raw data smoothing, background adjustment and normalization to in internal standard in HTP. Furthermore, glyXtool V3.0 includes an automated peak picking algorithm, a peak comparison function for biomarker screening and a peak annotation with access to a continuous growing oligosaccharide database. A graphic user interface makes this tailor made analysis tool also easy to handle. This novel modular glycoanalysis software-tool for data-processing and automated structural elucidation by interfacing a corresponding oligosaccharide-database (initiated and to be further built-up) will allow fully automated, highly sensitive instrument-, lab- and operator-independent high-throughput HTP-glycoanalysis, even when operated by non-experts. This is groundbreaking and in contrast to the currently prevailing methods, where multiplexing with respect to high-throughput is highly cost and lab-space intensive and ties up a lot of manpower and experts hands-on-time.
[1]Laroy, W.; Contreras, R.; Callewaert, N.; Glycome mapping on DNA sequencing equipment. Nature Protocols (2006) 1, 397 - 405.
[2]Schwarzer, J.; Rapp, E.; Reichl, U.; N-glycan analysis by CGE–LIF: Profiling influenza A virus hemagglutinin N-glycosylation during vaccine production. Electrophoresis (2008) 29, 4203 – 4214.
[3]Ruhaak, L.R.; Hennig, R.; Huhn, C.; Borowiak, M.; Dolhain, R. J. E. M.; Deelder, A. M.; Rapp, E.; Wuhrer, M.; Optimized workflow for preparation of APTS-labeled N-glycans allowing high-throughput analysis of
