In-depth Analysis Of The Synaptic Plasma Membrane Proteome Of Small Hippocampal Slices Using An Integrated Approach

BackgroundThe conventional approach generally leads to weak enrichment and contamination from other synaptic compartments such as the synaptic mitochondria and vesicles,and requires relatively high sample loads,which is probably a major disadvantage in this area of neuroscience research.The development of innovative approaches for studying the SPM proteome of small brain tissues is highly desired.ObjectiveThe aim of this study was to enrich the synaptic plasma membrane proteins for effective proteomic analysis.MethodsThis study presents a suitable protocol that integrates biotinylation-based affinity capture of cell surface-exposed proteins,isolation of synaptosomes,and biochemical extraction of SPM proteins from biotinylated hippocampal slices.After effectiveness evaluation of this integrated method by immunoblotting,we used highly sensitive mass spectrometry and streamlined bioinformatics to analyze the obtained SPM protein-enriched fraction.ResultsThe effectiveness of this integrated method was initially confirmed using immunoblot analysis of synaptic markers.Subsequently,we used highly sensitive mass spectrometry and streamlined bioinformatics to analyze the obtained SPM protein-enriched fraction.Our workflow positively identified 241 SPM proteins comprising 85 previously reported classical proteins from the pre-and/or post-synaptic membrane and 156 nonclassical proteins that localized to both the plasma membrane and synapse,and have not been previously reported as SPM proteins.Further analyses revealed considerable similarities in the physicochemical and functional properties of these proteins.Analysis of the interaction network using STRING indicated that the two groups showed a relatively strong functional correlation.Using MCODE analysis,we observed that 65 nonclassical SPM proteins formed 12 highly interconnected clusters with 47 classical SPM proteins,suggesting that they were the more likely SPM candidates.ConclusionTaken together,the results of this study provide an integrated tool for analyzing the SPM proteome of small brain tissues,as well as a dataset of putative novel SPM proteins to improve the understanding of hippocampal synaptic plasticity.