Human Milk Glycans: Isomeric Mixtures, Novel Structures, and MS~n

摘要

HMG samples were disassembled in the ion-trap to determine structural details. A combination of de novo sequencing and library matching approaches were used to determine topology and linkage, as well as definitive determination of interesting structural epitopes (Lewis structures, H antigens, etc.). Mass spectral data alone was able to determine Lewis A, X, and B structures, as well as H1 antigen structures. This work extends the library matching application previously presented where fragment spectra from standard materials can be used to assign structures to biological unknowns. Computationally generated similarity scores provide quantitative assessments of spectral matching, beyond visual data interpretation. In addition, MS~n data was useful for providing empirical information about isomeric mixtures and branching isomers. Permethylation of the HMGs provided direct evidence of branching patterns through the typical OH/OCH3 mass differences. This was key to several samples with isomers differing in lactose core branching. In contrast to lectin-binding studies, MS~n data also provided empirical evidence of unexpected branching isomers. As mentioned previously, permethylation and MS~n data could easily distinguish branching points on the core lactose compared to antennal lactosamine units. This revealed a tri-LacNAc motif that was expected at certain decaose isomers, but unexpected for some octaose structures. Internal fucose substitutions were also directly interrogated and assigned via MS~n; these were internal Lewis X structures, confirmed by MS~n but difficult to assign with lectins.