Proteomic comparison of different synaptosome preparation procedures

Abstract

Synaptosomes are frequently used research objects in neurobiology studies focusing on synaptic transmission as they mimic several aspects of the physiological synaptic functions. They contain the whole apparatus for neurotransmission, the presynaptic nerve ending with synaptic vesicles, synaptic mitochondria and often a segment of the postsynaptic membrane along with the postsynaptic density is attached to its outer surface. As being artificial functional organelles, synaptosomes are viable for several hours, retain their activity, membrane potential, and capable to store, release, and reuptake neurotransmitters. Synaptosomes are ideal subjects for proteomic analysis. The recently available separation and protein detection techniques can cope with the reduced complexity of the organelle and enable the simultaneous qualitative and quantitative analysis of thousands of proteins shaping the structural and functional characteristics of the synapse. Synaptosomes are formed during the homogenization of nervous tissue in the isoosmotic milieu and can be isolated from the homogenate by various approaches. Each enrichment method has its own benefits and drawbacks and there is not a single method that is optimal for all research purposes. For a proper proteomic experiment, it is desirable to preserve the native synaptic structure during the isolation procedure and keep the degree of contamination from other organelles or cell types as low as possible. In this article, we examined five synaptosome isolation methods from a proteomic point of view by the means of electron microscopy, Western blot, and liquid chromatography-mass spectrometry to compare their efficiency in the isolation of synaptosomes and depletion of contaminating subcellular structures. In our study, the different isolation procedures led to a largely overlapping pool of proteins with a fairly similar distribution of presynaptic, active zone, synaptic vesicle, and postsynaptic proteins; however, discrete differences were noticeable in individual postsynaptic proteins and in the number of identified transmembrane proteins. Much pronounced variance was observed in the degree of contamination with mitochondrial and glial structures. Therefore, we suggest that in selecting the appropriate isolation method for any neuroproteomics experiment carried out on synaptosomes, the degree and sort/source of contamination should be considered as a primary aspect.

The Effect of Sleep Deprivation and Subsequent Recovery Period on the Synaptic Proteome of Rat Cerebral Cortex

“…The applied procedure for synaptosome isolation is capable to enrich both the presynaptic and postsynaptic components of the synapses and to deplete the sample from extrasynaptosomal mitochondria and oligodenrocyte-derived structures. However, it contains a relatively high astrocyte-derived contamination [ 30 ]. Since there is an overlap in the protein expression profile of astrocytes and neurons, in the case of proteins present in both cell types, a detected quantitative alteration cannot be attributed to a given cell type.…”

“…Synaptosome enrichment was performed following the protocols of Phillips et al and Hahn et al with minor modifications [28][29][30]. Briefly, rats were deeply anesthetized with i.p.…”

An Shen Ding Zhi Ling Ameliorates the Symptoms of Attention Deficit Hyperactivity Disorder via Modulating Brain-Derived Neurotrophic Factor-Related Signaling Pathways

“…Synaptosomes can mimic various aspects of synaptic functions, which are continually used as objects in neurobiology studies [ 33 , 34 ]. The release of neurotransmitters is maintained through endocytosis and refilling of presynaptic local synaptic vesicles [ 35 ].…”

An Optimized Comparative Proteomic Approach as a Tool in Neurodegenerative Disease Research

“…In terms of extraction methods themselves, a number of studies have demonstrated enormous variability in proteome coverage merely based on the method of extraction, such as the use of a Dounce or motorised hand-held homogenizer versus a total tissue dissociation tool [ 39 , 40 ]. Different dissociation techniques and implementation of ice-cold temperatures must be particularly considered in preparations for non-neuronal cell types such as microglia, which due to high reactivity in the event of an “insult” such as enzymatic digestion and physical disruption, may exhibit a potentially confounding proteomic profile upon analysis [ 41 ].…”