Divergent immunomodulatory properties of milk proteins from Gannan yak, cattle-yak, and Holstein cattle: Insights from a comparative proteomics and murine model study.

While Gannan yak milk and Gannan cattle-yak milk are unique livestock resources in Gansu Province, China, the specific functional properties of their milk proteins remain to be fully elucidated. To address this, we employed tandem mass tag (TMT) quantitative proteomics to analyze the differentially expressed proteins (DEPs) among Gannan yak milk proteins (YMP), Gannan cattle-yak milk proteins (CYMP), and Holstein milk proteins (HMP). A total of 648 proteins were quantitatively analyzed. Compared with HMP, the DEPs in YMP and CYMP were primarily enriched in immune-related pathways such as the Complement and coagulation cascades, PPAR signaling pathway, and Phagosome. Conversely, the DEPs identified from the CYMP vs. YMP comparison were mainly associated with metabolic pathways, notably the Ribosome pathway. The immunomodulatory effects of these milk proteins were further evaluated in a cyclophosphamide (CTX)-induced immunosuppressed mouse model. Administration of YMP and CYMP significantly ameliorated weight loss, restored thymus/spleen indices, and enhanced serum immunoglobulins (IgG, IgM) and cytokines (IL-4, IL-6, TNF-α). They also reversed the imbalance in splenic T-lymphocyte subsets, increasing CD3+T and CD4+T cells while decreasing CD8+T cells, thereby normalizing the CD4+/CD8+ ratio. Although YMP and CYMP exhibited largely comparable overall efficacy, YMP consistently demonstrated a trend toward greater potency across multiple parameters. In contrast, HMP showed markedly weaker immunomodulatory activity. This research establishes a clear hierarchy in the immunomodulatory properties of these milk proteins, highlighting the potent and similar effects of YMP and CYMP, and provides a scientific basis for the high-value utilization of dairy resources in the Gannan region.