Deep and Precise Quantification of the Mouse Synaptosomal Proteome Reveals Substantial Remodeling during Postnatal Maturation

During
postnatal murine maturation, behavioral patterns emerge
and become shaped by experience-dependent adaptations. During the
same period, the morphology of dendritic spines, the morphological
correlates of excitatory synapses, is known to change, and there is
evidence of concurrent alterations of the synaptosomal protein machinery.
To obtain comprehensive and quantitative insights in the developmental
regulation of the proteome of synapses, we prepared cortical synaptosomal
fractions from a total of 16 individual juvenile and adult mouse brains
(age 3 or 8 weeks, respectively). We then applied peptide-based iTRAQ
labeling (four pools of 4 animals) and high-resolution two-dimensional
peptide fractionation (99 SCX fractions and 3 h reversed-phase gradients)
using a hybrid CID–HCD acquisition method on a Velos Orbitrap
mass spectrometer to identify a comprehensive set of synaptic proteins
and to quantify changes in protein expression. We obtained a data
set tracking expression levels of 3500 proteins mapping to 3427 NCBI
GeneIDs during development with complete quantification data available
for 3422 GeneIDs, which, to the best of our knowledge, constitutes
the deepest coverage of the synaptosome proteome to date. The inclusion
of biological replicates in a single mass spectrometry analysis demonstrated
both high reproducibility of our synaptosome preparation method as
well as high precision of our quantitative data (correlation coefficient R = 0.87 for the biological replicates). To evaluate the
validity of our data, the developmental regulation of eight proteins
identified in our analysis was confirmed independently using western
blotting. A gene ontology analysis confirmed the synaptosomal nature
of a large fraction of identified proteins. Of note, the set of the
most strongly regulated proteins revealed candidates involved in neurological
processes in health and disease states. This highlights the fact that
developmentally regulated proteins can play additional roles in neurological
disease processes. All data have been deposited to the ProteomeXchange
with identifier PXD000552.