Labeling and detecting human milk oligosaccharides through enzymatic incorporation of azido sialic acid and gel electrophoresis

Authors: Wu, Zhengliang; Naumann, Todd; Bowman, Michael; Skory, Christopher

Submitted to: Glycobiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/11/2026
Publication Date: 4/21/2026
Citation: Wu, Z.L., Naumann, T.A., Bowman, M.J., Skory, C.D. 2026. Labeling and detecting human milk oligosaccharides through enzymatic incorporation of azido sialic acid and gel electrophoresis. Glycobiology. https://doi.org/10.1093/glycob/cwag030.
DOI: https://doi.org/10.1093/glycob/cwag030

Interpretive Summary: Human milk oligosaccharides (HMOs) are complex sugars found in breast milk that play an important role in in infant development. They promote the growth of beneficial bacteria in the gut, prevent growth of pathogens, and stimulate immune system development. ARS researchers in Peoria, Illinois, are investigating methods to manufacture HMOs from agricultural sugars so they can be used to supplement infant formulas. As part of this work, they developed a novel, easy to use procedure to analyze HMOs using fluorescent detection methods. This technique can quickly identify the presence of HMOs and provide useful information about their structure without the need for expensive analytical equipment. This work will facilitate discovery of efficient production methods for HMOs and provide new value-added bioproducts for agricultural sugars.

Technical Abstract: Human milk oligosaccharides (HMOs) are complex sugars present in human milk. These sugars possess prebiotic, immunomodulatory, and antagonistic properties towards pathogens and therefore are important for the health and well-being of newborn babies. Here we demonstrate the detection of certain HMOs using enzymatic incorporation of azido sialic acid, especially those neutral core HMOs with a non-reducing end N-acetyllactosamine (Gal-beta-1,4-GlcNAc) unit. Neutral core HMOs are considered as the precursors for all HMOs and contain unmodified N-acetyllactosamine or lacto-N-biose (Gal-beta-1,3-GlcNAc) at their nonreducing ends. Azido sialic acid is first enzymatically introduced to these HMOs via ST6 beta-galactoside a-2,6-sialyltransferase 1 (ST6Gal1). The tagged HMOs are then clicked to DBCO-Cy5 through copper-free azide-alkyne cycloaddition and separated by polyacrylamide gel electrophoresis and visualized with fluorescent imaging. As demonstration, HMOs were detected in human and bovine milk whey preparations as well as in purified HMO fractions. The detected HMO ranged from disaccharides to decasaccharides as suggested by HMO standards and mass spectrometry analysis. The data also suggested the presence of polysaccharides in human milk. In contrast, no equivalent oligosaccharides were detected in bovine milk samples. The results clearly demonstrated the uniqueness of HMOs. The method offers a convenient way for profiling these HMOs in human milk or baby formula.