Mapping of the synaptic mitochondrial interactome by crosslinking mass spectrometry (XL-MS)

Neuronal communication is based on synaptic signal transmission with neurotransmitter release from synaptic vesicles at presynaptic compartments upon stimulation of the synapse. The complex molecular processes involved in synaptic signal transmission are subject to various studies, yet they are still not fully understood. Furthermore, it has not yet been established in which ways the high energy levels necessary for these processes are provided. High numbers of mitochondria, which are responsible for generation of the vast majority of cellular energy among various other functions, are also present in distal axons and presynaptic compartments. However, relatively few studies have been dedicated to the role of mitochondria in neuronal cells and especially at the synapse. The aim of this study is to provide an unbiased, deep mapping of the synaptic mitochondrial protein interactome using chemical crosslinking mass spectrometry (XL-MS).

Chemical crosslinking of proteins is based on the formation of covalent bonds between amino acid residues in close spatial proximity upon reaction with the crosslinker. Subsequent MS analysis of endo-proteolytically digested protein samples results in identification of the crosslinked amino acid residues and their position within the peptide or protein. This provides insight into protein interactions based on the spatial restraints of crosslinking reactions.

We performed chemical crosslinking under near-physiological conditions on synaptosomes (pinched-off synaptic terminals), which are common model systems for the study of synaptic functions. Synaptosomes were isolated and purified from rat brain tissue, followed by crosslinking and further subfractionation of crosslinked synaptosomes with enrichment for (intra-synaptosomal) mitochondria. LC-MS acquisition was performed upon proteolytically digested peptide samples pre-fractionated with reverse-phase C18 chromatography at basic pH. The XL-MS workflow was systematically optimized to maximize the number of identified inter-protein crosslinks. As results obtained by XL-MS studies are still limited by the complex computational analysis of MS acquisition files for crosslink identifications, two crosslinkers (disuccinimidyl suberate (DSS) and disuccinimidyl sulfoxide (DSSO)) with different properties and different subsequent search strategies were applied in this study and compared for quantity and quality of crosslink identifications. The resulting inventory of inter-crosslinks of mitochondrial proteins, as well as between mitochondrial and other synaptosomal proteins, were visualized in an interaction map, which can hopefully provide the basis for follow-up experiments to explore selected protein-protein interactions of mitochondria at the synapse.