Supplementary Material for: Proteomic Analysis of Mouse Cortex Postsynaptic Density following Neonatal Brain Hypoxia-Ischemia

Proteomics of the synapses and postsynaptic densities (PSDs) have
provided a deep understanding of protein composition and signal networks
in the adult brain, which underlie neuronal plasticity and
neurodegenerative or psychiatric disorders. However, there is a paucity
of knowledge about the architecture and organization of PSDs in the
immature brain, and how it is modified by brain injury in an early
developing stage. Mass spectrometry (MS)-based proteomic analysis was
performed on PSDs prepared from cortices of postnatal day 9 naïve mice
or pups which had suffered hypoxic-ischemic (HI) brain injury. 512
proteins of different functional groups were identified from PSDs
collected 1 h after HI injury, among which 60 have not been reported
previously. Seven newly identified proteins involved in neural
development were highlighted. HI injury increased the yield of PSDs at
early time points upon reperfusion, and multiple proteins were recruited
into PSDs following the insult. Quantitative analysis was performed
using spectral counting, and proteins whose relative expression was more
than 50% up- or downregulated compared to the sham animals 1 h after HI
insult were reported. Validation with Western blotting demonstrated
changes in expression and phosphorylation of the N-methyl-D-aspartate
receptor, activation of a series of postsynaptic protein kinases and
dysregulation of scaffold and adaptor proteins in response to neonatal
HI insult. This work, along with other recent studies of synaptic
protein profiling in the immature brain, builds a foundation for future
investigation on the molecular mechanisms underlying developing
plasticity. Furthermore, it provides insights into the biochemical
changes of PSDs following early brain hypoxia-ischemia, which is helpful
for understanding not only the injury mechanisms, but also the process
of repair or replenishment of neuronal circuits during recovery from
brain damage.