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 delivery 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 early lactation (first 10 days postpartum) and during later lactation (week five postpartum). 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. On average, a total of 662 proteins were identified per sample, of which 169 were consistently quantified across all samples (1% FDR). During early lactation, 10 proteins differed significantly, with bidirectional changes and moderate effect sizes. At week five, 19 proteins were differentially abundant, predominantly higher in preterm samples. Immune-related proteins constituted the largest functional category at both stages. Immunoglobulin heavy constant gamma 4 remained consistently downregulated in preterm milk (1.6-fold lower abundance). Ferritin heavy chain (1.5) and HLA class II histocompatibility antigen gamma chain (1.8) were elevated only early, whereas calprotectin subunits S100A8 (5.6) and S100A9 (5.2) were markedly upregulated later. Conclusions: Proteomic differences vary across lactation stages, highlighting lactation stage as an essential contextual variable in comparative milk proteomics.