Time-Dependent Differences in the Human Milk Proteome After Preterm Birth: A Paired Two-Stage Proteomic Study

Background: /Objectives: Human milk composition is shaped by gestational age at de-livery and stage of lactation; however, proteomic differences between milk from mothers of preterm and term infants and their temporal patterns remain incompletely characterised. Methods: This prospective study enrolled 40 lactating mothers: 20 who delivered preterm infants (< 32 weeks’ gestation) and 20 who delivered at term (37–42 weeks). Each provided milk samples during first 10 days postpartum and at the fifth week. Milk serum was analysed using quantitative data-independent acquisition mass spectrometry. Differential protein abundance was assessed separately at each time point; functional annotation was performed using Gene Ontology biological process analysis. Results: Eighty samples were analysed. During early lactation, 10 proteins differed significantly, most showing lower abundance in preterm milk. At week five, 19 pro-teins were differentially abundant, predominantly higher in preterm samples. Im-mune-related proteins constituted the largest functional category at both stages. Im-munoglobulin heavy constant gamma 4 remained consistently downregulated in pre-term milk (fold change −1.6). Ferritin heavy chain (1.5) and HLA class II histocompati-bility antigen gamma chain (1.8) were elevated only early, whereas calprotectin subu-nits S100A8 (5.6) and S100A9 (5.2) were markedly upregulated later. Conclusions: Proteomic differences vary across lactation stages, identifying lactation timing as a critical contextual factor in comparative human milk proteome studies.