Lactation-stage specific protein shifts in koala milk mirror the joey's growth needs

Koalas (Phascolarctos cinereus) give birth to highly altricial young that rely on maternal milk for nutritional support and immune protection in the first year of life. This study applied proteomics to analyse stage-specific shifts in the koala milk proteome across early-, mid-, and late-lactation. Proteins were extracted from seven samples and analysed using liquid chromatography-tandem mass spectrometry. Quantitative profiling identified 874, 1220, and 498 proteins in early-, mid- and late-lactation stages, respectively. The most abundant proteins-including β-lactoglobulin, caseins, clusterin, trichosurin, albumin and lactotransferrin-were consistently detected across all stages, reflecting crucial roles in nutrition, immune function, and detoxification. Protein-protein interaction analyses revealed functionally important protein clusters associated with energy metabolism, translation, protein transportation, and these clusters exhibited shifts in abundance that align with the developmental and metabolic demands of the joey. Gene set variation analysis revealed stage-dependent regulation of immune pathway proteins, supporting foundational immune development in early-lactation, heightened neuroimmune protection in mid-lactation, and increased immune competence in late-lactation. These findings provide comprehensive insight into the dynamic adaptation of koala milk composition to joey development and may inform the design of nutritionally and immunologically optimised milk replacers for koala conservation and rehabilitation.