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要旨:
Glycomics is a rapidly emerging field that can be viewed as a complement to other „omics“ approaches including proteomics and genomics. 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, fully automated high-throughput (HTP) and high-resolution (HR) analysis methods including automated data evaluation are required.
Besides several mass spectrometry and liquid chromatography based analysis techniques, electromigrative separation techniques for the analysis of oligosaccharides have been developed over the last years. Especially, capillary gel electrophoresis with laser induced fluorescence detection (CGE-LIF) shows high potential for HTP glycoprofiling of fluorescently labeled glycans [1,2]. This glycoanalysis approach based on multiplexed CGE-LIF is utilizing a DNA-sequencer. The instruments - based electromigrative separation - in principle (with some minor modifications) are well suited for glyco-profiling of glycoconjugates.
The aim of the project presented was to further investigate and to improve this innovative approach with respect to sample preparation and data analysis. First, sample preparation method and workflow were further optimized with respect to performance and feasibility regarding HTP. Second, data analysis was computerized developing a novel modular software-tool for automated data-processing and structural elucidation by interfacing a corresponding oligosaccharide-database. Using this tool, the generated “normalized” electropherograms of the glyco-moieties ("fingerprints") can be evaluated on two stages: simple qualitative and quantitative fingerprint comparison and structural elucidation of each single glyco-component. 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].
This novel modular glycoanalysis system and method allows fully automated, highly sensitive instrument-, lab- and operator-independent high-throughput HTP-glycoanalysis, even when operated by non-experts. This is 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]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.
[2]Laroy W, Contreras R, Callewaert N: Glycome mapping on DNA sequencing equipment. Nature Protocols (2006) 1, 397 - 405.
[3]Ruhaak LR, Hennig R, Huhn C, Borowiak M, Dolhain RJEM, Deelder AM, Rapp E, Wuhrer M: Optimized workflow for preparation of APTS-labeled N-glycans allowing high-throughput analysis of human plasma glycomes using 48-channel multiplexed CGE-LIF. Journal Prot. Res. (2010) 9, 6655 – 6664.
