Datensatz

Zusammenfassung

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 increase in the demand for sophisticated databases and analytical tools in glycobiology, respectively glycobiotechnology. In order to enhance and improve the comparatively small set of existing glyco-analytical methods and toolboxes, a system and method for automated straightforward, sensitive high-throughput (HTP) and high-resolution glycoanalysis was developed [1,2]. This glycoanalysis approach is based on multiplexed capillary gelelectrophoresis with laser induced fluorescence detection (CGE-LIF), using a DNA-sequencer (Applied Biosystems, USA). Data is evaluated in conjunction with a novel modular software-tool for data-processing and automated structural elucidation by interfacing a corresponding oligosaccharide-database (initiated and to be further built-up). The aim of the project presented was to further investigate and to improve this innovative method with respect to sample preparation and sensitivity. Therefore, the sample preparation method and the workflow were further optimized with respect to its performance and feasibility regarding HTP. In addition, the limit of detection (LOD) was determined. An improved post-labeling sample clean-up via size exclusion chromatography (SEC) compared to [1,3] is shown. The replacement of time- consuming consecutive CGE-LIF analyses of the SEC-fractions before pooling by a HTP-ready fluorescence assay, using a 96-wellplate reader is demonstrated. The optimized SEC protocol is faster and shows higher clean-up power as the established method . Furthermore, the new set-up in 96 well format together with the newly established time-saving fluorescence assay enables higher throughput in sample preparation. And finally, the LOD for fluorescently labeled oligosaccharides turned out to be in the lower attomole range down to zeptomoles regarding the absolute amount injected. [1]Schwarzer, J.; E. Rapp; U. Reichl; N-glycan analysis by CGE-LIF: Profiling influenza A virus hemagglutinin N-glycosylation during vaccine production. Electrophoresis (2008) 29 4203-4214. [2]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 human plasma glycomes using 48-channel multiplexed CGE-LIF. Journal of Proteome Research (2010) 9, 6655 – 6664. [3]Laroy, W.; Contreras, R.; Callewaert, N.; Glycome mapping on DNA sequencing equipment. Nature Protocols (2006) 1, 397 - 405.