Robert Kottler

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

During the last decade, enormous progress regarding knowledge about composition and properties of human milk (HM) has been made. Besides nutrition, the three macro‐nutrients: proteins, lipids, and carbohydrates combine a large variety of properties and functions. Especially, complex oligosaccharides emerge as important dietary factors during early life with multiple functions. The characterization of these HM oligosaccharides (HMOS) within the total carbohydrate fraction is prerequisite to understand the relationship between milk composition and biological effects. Therefore, extended studies of large donor cohorts and thus, new high‐throughput glycoanalytical methods are needed. The developed method comprises sample preparation, as well as analysis of HMOS by multiplexed CGE with LIF detection (xCGE‐LIF). Via a respective database the generated “fingerprints” (normalized electropherograms) could be used for structural elucidation of HMOS. The method was tested on HM samples from five different donors, partly sampled as a series of lactation time points. HMOS could be easily identified and quantified. Consequently, secretor and Lewis status of the donors could be determined, milk typing could be performed and quantitative changes could be monitored along lactation time course. The developed xCGE‐LIF based “real” high‐throughput HMOS analysis method enables qualitative and quantitative high‐performance profiling of the total carbohydrate fraction composition of large sets of samples.

N -Glycosylation Fingerprinting of Viral Glycoproteins by xCGE-LIF

The ongoing threat of pathogens, increasing resistance against antibiotics, and the risk of fast spreading of infectious diseases in a global community resulted in an intensified development of vaccines. Antigens used for vaccination comprise a wide variety of macromolecules including glycoproteins, lipopolysaccharides, and complex carbohydrates. For all of these antigens the sugar composition plays a crucial role for immunogenicity and protective efficacy of the vaccine. Here, we provide a protocol for N-glycosylation fingerprinting utilizing high performance multiplexed capillary gel electrophoresis with laser-induced fluorescence detection (xCGE-LIF) technology. The method described, enables to analyze the N-glycosylation of specific proteins out of a complex sample or even the total of all N-glycans contained in such a sample. The protocol is exemplarily demonstrated for N-glycosylation fingerprinting of cell culture-derived influenza A and B viruses and their major antigens, the membrane glycoproteins hemagglutinin and neuraminidase.