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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 glyco-analytical toolbox, fully automated high-throughput (HTP) and high-resolution analysis methods including automated data evaluation are required. Especially one glycoanalysis approach, based on multiplexed capillary gel electrophoresis with laser induced fluorescence detection (CGE-LIF) utilizing a DNA-sequencer, shows high potential for HTP glycoprofiling of glycoconjugates 1,2,3.
The aim of the project presented was to further investigate and to improve this innovative approach for different fields of application with respect to sample preparation, separation and data analysis. First, sample preparation method and workflow were further optimized with respect to performance and feasibility regarding HTP. Second, with up to 96 capillaries in parallel, the fully automated separation with an impressive sensitivity is shown to result in massive reduction of the effective separation time per sample. Third, data analysis was automated developing a novel modular software-tool for data-processing and -analysis, interfacing a corresponding oligosaccharide-database. Using this software-tool, the generated “normalized” electropherograms of glycomoieties (“fingerprints”) can be evaluated on two stages: “simple” qualitative and quantitative fingerprint comparison and structural elucidation of each single glycocomponent. The smart applicability of this technique is demonstrated for different types of glycosamples such as the “glycome” of (monoclonal) antibodies, human milk and of human blood serum. 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.
References
1.Schwarzer J, Rapp E, Reichl U, Electrophoresis, 2008, 29, 4203-4214.
2.Laroy W, Contreras R, Callewaert N, Nature Protocols, 2006, 1, 397-405.
3.Ruhaak LR, Hennig R, Huhn C, Borowiak M, Dolhain RJEM, Deelder AM, Rapp E, Wuhrer M, Journal of Proteome Research, 2010, 9, 6655-6664.
