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.

Keywords: Neuroproteomics; Proteomics; Subcellular proteomics; Synapse; Synaptosome.

Fig. 1

Validation and morphometric analysis of the purity of synaptosomal preparations with electron microscopy. a–e) Representative electron micrographs and the histograms of the synaptosomal size distribution are shown for all fractionation methods. f and f’ The left panel (f) shows an irregular-shaped multi-membranous structure in #5 Method preparation at low magnification and the right panel (f’) shows its magnified part. g and g’ Overview of the morphometric method. The left panel (g) shows the selection of the distinct components from an electron micrograph (green, certainly synaptosomes; blue, most likely synaptosomes; red, non-synaptosomal structures), while the one on the right-hand side (g’) presents colorized areas covered by the respective components. h Comparative analysis results on the areas of the subcellular components of the different synaptosomal preparations. **0.001 < p < 0.01; ***p < 0.001 (post hoc Mann–Whitney tests). Means ± SEM are shown for pooled data (12 images per sample) of each preparation. Scale bars: a–e, g: 0.5 µm, f: 1 µm, f’: 50 nm

Fig. 2

Western blot analysis of different synaptosomes preparations. Immunopositive bands and densitometric analysis are shown for synaptic- (a, b), glial- (c, d) and mitochondrial (e, f) marker proteins. Densitometric values in each sample are shown after normalization to the signal detected in the unfractionated cortical homogenate

Fig. 3

The numbers of common and unique proteins identified by LC–MS/MS in the synaptosome samples