Every hour counts: mapping neonatal blood plasma proteome changes during the first 2 days of life at hourly resolution

Birth represents a dramatic shift from a quasi-sterile intrauterine life to an antigen-rich extrauterine environment. The transition from the fetal state with induced maternal tolerance to a state where new immune challenges must be met, necessitates a significant immune system adaptation. The main components of the immune system circulate in plasma. Given the plasma"s major immune modulatory role, these adaptations are reflected by changes to the plasma proteome composition. We collected 906 peripheral blood samples from 679 healthy term newborns at day of life 0 and day of life 1 as part of an ongoing newborn vaccination study. Carefully recording the time of birth and time of blood collection allowed us to calculate the exact age of the newborn in hours, rather than the commonly applied daily resolution. We used a multiplexed antibody-based assay for monitoring cytokines and chemokines, and a targeted liquid chromatography-mass spectrometry assay for monitoring the plasma proteome. We utilized a sample-sparing strategy, which was a prerequisite for this study given the limited nature of newborn blood sample volumes. This 2-pronged strategy allowed us to monitor the postnatal dynamics of nearly 400 proteinaceous analytes in plasma during the first 48 hours of life at an hourly resolution, with a throughput of ~100 samples per day. The analysis unveiled distinct and dynamic changes in developmental trajectories of the proteome. The detailed study of these crucial first 48 hours post birth at an hourly resolution provided more accurate information about fold changes and dynamics, insights into the ordering of protein changes, and co-regulations – all these findings could not be gleaned from the conventional time course analysis at day resolution, which compress complex dynamics over 24 hours into a single measurement with large error bars. Notable observations included 10-fold increases of haptoglobin during the first 24 hours of life and complex dynamics for CXCL8, with little net change at conventional day resolution. This first-of-its-kind high resolution plasma proteome ontogeny study delineated postnatal immune system development in healthy newborns immediately after birth, and mapped the temporal molecular roadmap including several critical periods during the first 48 hours of life, which possibly demarcate important postnatal physiological processes such as the postnatal closure of the ductus arteriosus, which bypasses the liquid-filled non-functioning lungs in fetuses.