GlycoBond X enables isomer-resolved milk oligosaccharide glycomics and reveals evolutionary divergence across four mammalian species.

Milk oligosaccharides (MOs) are essential for the development of mammalian offspring, yet their fine-scale structural evolutionary divergence remains unelucidated, largely due to isomeric complexity and the limitations of analytical methods. Here, we present GlycoBond X, an online platform that couples high-resolution separation with parallel structural characterization of glycan isomers through multi-stage chemical derivatization and RP-HPLC-MS/MS. By applying this strategy to four evolutionarily distinct mammals, we uncovered a conserved transition from acidic MO dominance (>75% in mouse and tree shrew) to predominantly fucosylated neutral MOs (>62% in macaque and human). GlycoBond X unveiled unprecedented structural diversity, including 22 unique fucosylation motifs and 66 previously undescribed human MOs. Notably, tree shrew MOs exhibited human-like structures and shared over 69% FUT2 sequence homology with primates. This study established a high-throughput, high-sensitivity platform, elucidated the adaptive structural evolution of oligosaccharides via evolutionary glycomics, and provided a foundation for exploring their biosynthetic pathways.