Abstract
During the last decade enormous progress regarding knowledge about the composition and properties of (human) milk has been made. The three main fractions of milk - proteins, fats and carbohydrates - combine a large variety of properties and functions, like lowering the risk of infections or balancing/teaching the developing immune system of newborns. In the recent past especially complex sugar structures, such as free oligosaccharides from human milk (HMOS), animal milk and other sources became of increasing interest. Their investigation gained importance, e.g.: in clinical research and the development and production of functional food and food additives. The work presented, comprises the establishment and improvement of a high-performance glycoanalysis technique, based on multiplexed capillary gel electrophoresis with laser induced fluorescence detection (xCGE-LIF), that allows for high-throughput (HT) characterization of HMOS [1, 2]. The resulting rapid and sensitive method for HT-analysis of the HMOS fraction composition, using a CGE-LIF based multiplexing capillary DNA sequencer, includes sample preparation steps like, protein-precipitation, clean-up, derivatization as well as the use of the automated glycoanalysis software “glyXtool”. In combination with a dedicated HMOS database, which was built-up in-house, the generated HMOS “fingerprints” (normalized electropherograms) can be used for in-depth glycoanalysis, i.e.: structural elucidation of each single HMOS compound and its relative quantification. The robustness of the developed method was tested on various human milk samples (time series of different donors). Several factors were identified to be responsible for differences in HMOS-composition, including origin of the mother, lactation time point and genetic determinants (secretor-gene and lewis-gene). The combination of these two antigenic determinants defines four human milk types [3]. It turned out, that xCGE-LIF is a valuable alternative to the existing time consuming analysis techniques. The developed method allows straightforward, fast, unequivocal and sensitive analysis of HMOS fraction composition, including an easy determination of the secretor and lewis status of milk donors. [1] Schwarzer, J.; Rapp, E.; Reichl, U.; N-Glycan Analysis by Capillary Gel Electrophoresis - Profiling Influenza A Virus Hemagglutinin N-Glycosylation during Vaccine Production. Electrophoresis (2008), 29, 4203-4214. [2] 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. Glycoconjugate Journal (2011), 28(5), 330-331. [3] Thurl, S.; Munzert, M.; Henker, J.; Boehm, G.; Müller-Werner, B.; Jelinek, J.; Stahl, B.; Variation of human milk oligosaccharides in relation to milk groups and lactational periods. British Journal of Nutrition (2010), 104, 1261-1271.
