Structural characterization of human milk oligosaccharides using ultrahigh performance liquid chromatography-helium charge transfer dissociation mass spectrometry

The combination of helium charge transfer dissociation mass spectrometry (He-CTD-MS) with ultrahigh performance liquid chromatography (UHPLC) is presented for the analysis of a complex mixture of acidic and neutral human milk oligosaccharides (HMOs). The research focuses on the identification of the monosaccharide sequence, the branching patterns, the sialylation/fucosylation arrangements, and the differentiation of isomeric oligosaccharides in the mixture. Initial studies first optimized the conditions for the UHPLC separation and the He-CTD-MS conditions. Results demonstrate that He-CTD is compatible with UHPLC timescales and provides unambiguous glycosidic and cross-ring cleavages from both the reducing and the nonreducing ends, which is not typically possible using collision-induced dissociation. He-CTD produces informative fragments, including (0,3)A(n) and (0,4)A(n) ions, which have been observed with electron transfer dissociation, electron detachment dissociation, and ultraviolet photodissociation (UVPD) and are crucial for differentiating the alpha-2,3- versus alpha-2,6-linked sialic acid (Neu5Ac) residues present among sialyllacto-N-tetraose HMOs. In addition to the linkage positions, He-CTD is able to differentiate structural isomers for both sialyllacto-N-tetraoses and lacto-N-fucopentaoses structures by providing unique, unambiguous cross-ring cleavages of types (0,2)A(n), X-0,2(n), and (1,5)A(n) while preserving most of the labile Neu5Ac and fucose groups.

Automated summary

The combination of helium charge transfer dissociation mass spectrometry with ultrahigh performance liquid chromatography provides a powerful tool for the analysis of complex mixtures of human milk oligosaccharides. This method allows for the identification of monosaccharide sequence, branching patterns, sialylation/fucosylation arrangements, and differentiation of isomeric oligosaccharides. The results demonstrate the compatibility of helium charge transfer dissociation with ultrahigh performance liquid chromatography and its ability to provide informative fragments for differentiation of alpha-2,3- versus alpha-2,6-linked sialic acid residues and differentiation of structural isomers.