The abundant proteins in human milk have been well characterized and are known to provide nutritional, protective, and developmental advantages to both term and preterm infants. However, relatively little is known about the expression of the low abundance proteins that are present in human milk because of the technical difficulties associated with their detection. We used a combination of electrophoretic techniques, ProteoMiner treatment, and two-dimensional liquid chromatography to examine the proteome of human skim milk expressed between 7 and 28 days postpartum by healthy term mothers and identified 415 in a pooled milk sample. Of these, 261 were found in human skim milk for the first time, greatly expanding our understanding of the human skim milk proteome. The majority of the proteins identified were involved in either the immune response (24%) or in cellular (28%) or protein (16%) metabolism. We also used iTRAQ analysis to examine the effects of premature delivery on milk protein composition. Differences in protein expression between pooled milk from mothers delivering at term (38-41 weeks gestation) and preterm (28-32 weeks gestation) were investigated, with 55 proteins found to be differentially expressed with at least 90% confidence. Twenty-eight proteins were present at higher levels in preterm milk, and 27 were present at higher levels in term milk.