Abstract
Carbohydrates are some of the most common biomolecules in nature. Being nearly ubiquitous present, in their simplest form they are acting as a universal energy source. Furthermore, these carbohydrates forming various sets of complex linear or branched homo- and hetero-oligo/polymers. E.g. they are building a big part of the extracellular matrix or alter protein properties as posttranslational modifications. For that reason carbohydrate analysis became of increasing importance in the recent past. To enhance the knowledge in glycobiology, respectively glycobiotechnology – “high-performance” glycoanalytical methods are required that allow in-depth glycoanalysis on large sample sets in an acceptable time.
Using mass spectrometry and liquid chromatography in various setups, as well as electrokinetic separation techniques, detailed information on glycosylation can be obtained. Especially, multiplexed capillary gel electrophoresis with laser induced fluorescence detection (xCGE-LIF) was recently established for “real” HT-profiling of APTS labeled N-glycans from glycoproteins in complex samples [1,2,3]. By using standard DNA sequencing equipment with up to 96 capillaries in parallel, a massive reduction of the effective separation time per sample can be achieved.
To ensure its HT-character, xCGE-LIF requires a fast and flexible sample preparation, optimized for ease of use. For that reason we developed a modular sample preparation toolbox with respect to the different needs, caused by the unique characteristics of each type of sample.
Starting with optional protein enrichment and/or protein separation, enzymatic glycan-release has to be performed, followed by glycan labeling with a fluorescent dye. Before xCGE-LIF measurement, optionally, post-labeling sample clean-up can be applied, based on size exclusion chromatography or hydrophilic interaction solid phase extraction. By using this sample preparation toolbox glycoanalysis of complex samples like citrate plasma, human milk oligosaccharides (HMOS), as well as glycoanalysis of more modest samples like Immunoglobuline G (IgG), Erythropoetin (EPO) and the influenza virus antigen Hemagglutinin is feasible with a minimal hands-on time.
The presented xCGE-LIF based glycoanalysis method, in combination with an automated data-processing via “glyXtool”, enables in-depth and “real” HT-analysis of complex carbohydrates like glycans.
[1]Laroy, W.; Contreras, R.; Callewaert, N.; Glycome mapping on DNA sequencing equipment. Nature Protocols (2006) 1, 397 - 405.
[2]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.
[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 human plasma glycomes using 48-channel multiplexed CGE-LIF. Journal of Proteome Research (2010) 9, 6655 – 6664.
