Milk is the primary source of nutrition for infant mammals during their early developmental stages, providing essential nutrients for growth, brain development, and immune system maturation. The Tammar wallaby’s (Macropus eugenii) lactation period spans 300-350 days following a short 26-day gestational period, during which the joey develops in the mother's pouch. This prolonged lactation underscores the critical role of milk in the survival and growth of the infant wallaby. Milk is a complex mixture of biological macromolecules, including proteins, lipids, and, predominantly, milk oligosaccharides. Importantly, proteins and lipids are also conjugated with oligosaccharides (glycans). These glycans undergo changes in composition and concentration to meet the evolving needs of the infant. While previous studies have characterized the N- and O-linked glycosylation patterns and proteome of Tammar wallaby milk, other major glycoconjugates have not been thoroughly explored.
This study expanded the Same Sample Sequential Multi-Glycomics (SSSMuG) workflow to include free oligosaccharides before analysis of other glycoconjugate classes (glycosaminoglycans, glycosphingolipid glycans, N- and O-linked glycans) along with proteomics. Glycans were enzymatically or chemically released from single, immobilised, 10 µL milk samples and analysed using high performance liquid chromatography coupled with fluorescence detection or mass spectrometry-based techniques. Results revealed significant variations in glycoconjugate classes over the lactation period, with oligosaccharides and glycosaminoglycans showing decreased heterogeneity, while glycosphingolipids remained stable. Additionally, N- and O-linked glycan patterns were consistent with previous findings, although new low-abundance structural isomers were identified potentially due to greater coverage from increased PNGase F enzyme function after glycosaminoglycan release. This study demonstrates that SSSMuG provides enhanced glycan and proteomic coverage from small amounts of milk samples, offering deeper insights into the changes in the glycoconjugates over lactation. These findings highlight the effectiveness of this approach in expanding our understanding of milk composition and its role in infant development.