High throughput characterization of the human milk oligosaccharide composition via multiplexed capillary gel electrophoresis with laser induced fluorescence detection

During the last decade an enormous progress regarding knowledge about the composition and properties of human milk (HM) has been made. The three main components of human milk are proteins, fats and carbohydrates. They combine a large variety of properties and functions, like lowering the risk of infections or stimulate/teach the developing immune system of newborns. The project presented describes the establishment and improvement of analytical techniques, which allow the high-throughput (HTP) characterization of human milk oligosaccharides (HMOS). The HMOS fraction consists primarily of lactose and a large variety of neutral and acidic oligosaccharides. Until now more than 100 of these HMOS have been detected via different analytical techniques based, e.g. on liquid chromatography, mass spectrometry, nuclear magnetic resonance spectroscopy. 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 still required. Eventually, this information can be used to improve the quality of infant nutrition. Therefore, a rapid and sensitive HTP-method for the analysis of the composition of the oligosaccharides (OS) in human milk was established. The method comprises protein-precipitation, OS purification, clean-up steps, OS derivatization with a fluorescent tag as well as a “fingerprint” analysis of OS pools by capillary gel electrophoresis with laser induced fluorescence detection (CGE-LIF) using a ABI PRISM 3100 capillary DNA-sequencer (Applied Biosystems, USA). The method developed was tested on various HM samples (time series of different donors). Several factors were identified to be responsible for a different HMOS-composition. These include the origin of the mother, the lactation time point and genetic determinants. Some of these factors affect the relative abundances along the lactation period, while others affect in principle the presence of an oligosaccharide, e.g. the antigenic determinants secretor-gene (Se) and lewis-gene (Le, determining the Lewis blood group system). Based on the combination of these two antigenic determinants four human milk types exist [1]. It turned out, that multiplexed CGE-LIF is a valuable alternative to the time consuming analysis techniques mentioned above. The method and system developed [2], allows a straightforward, fast, unequivocal and sensitive analysis of the oligosaccharide composition in human milk (the secondary gene products of the secretor-gene - respectively lewis-gene). Thereby, secretor and lewis status of the milk donor can be determined. [1]Thurl, S. et al., Variation of human milk oligosaccharides in relation to milk groups and lactational periods. British Journal of Nutrition (2010) 104, 1261-1271. [2]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