Method Development for Multiplexing CGE-LIF Based Glycoanalysis

Elucidation of the Carbohydrate Composition of Human Milk

Another project utilizing this glycoanalysis approach is the HT characterization of human milk oligosaccharides (HMOS) with respect to the improvement of artificial, respectively, functional food. The project was realized in cooperation with Nutricia Research/Danone, Human Milk Research in Utrecht/Netherlands. The HMOS fraction primarily consists of lactose and a large variety of neutral and acidic oligosaccharides. The characterization of HMOS is essential to understand the relationship between structure and biological effect. However, as the role and significance of HMOS is still not fully understood, identification and characterization of oligosaccharide structures is required.

Human Oligosaccharide (HMOS) "fingerprint" (normalized electropherogram) of the total carbohydrate fraction of different human milk types with peak annotation using the growing in-house HMOS database.

Multiplexed capillary gelelectrophoresis with laser induced fluorescence detection (xCGE-LIF), allowed the generation of HMOS "fingerprints" (normalized electropherograms). In combination with an in-house HMOS database, the system was used for in-depth glycoanalysis, i.e.: structural elucidation of each single compound and its relative quantification. The method developed was tested on various human milk samples (time series of different donors). Several factors were identified to be responsible for differences in HMOS-composition. These included the origin of the mother, the lactation time point, and genetic determinants, e.g.: the secretor-gene (Se) and the Lewis blood group system with the Lewis gene (Le).

Overall, the xCGE-LIF based HT glycoanalysis system enabled qualitative and quantitative glycoanalysis of large donor cohorts, and of cohort time series along the course of lactation.

Publications

Kottler, R.; Hennig, R.; Mank, M.; Stahl, B.; Reichl, U.; Rapp, E.: High-Throughput Characterization of the Human Milk Oligosaccharide Composition Utilizing a Multiplexing Capillary DNA Sequencer. In Glycoconjugate Journal, 28 (5 - Special Issue : XXI International Symposium on Glycoconjugates), p. 330. 21st International Symposium on Glycoconjugates (Glyco 21), Wien, Östereich, August 21, 2011 - August 26, 2011. (2011)
Kottler, R.; Mank, M.; Hennig, R.; Müller-Werner, B.; Stahl, B.; Reichl, U.; Rapp, E.: Development of a high-throughput glycoanalysis method for the characterization of oligosaccharides in human milk utilizing multiplexed capillary gel electrophoresis with laser-induced fluorescence detection. Electrophoresis 34 (16), pp. 2323 - 2336 (2013)
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