Hidden proteome of synaptic vesicles in the mammalian brain

Current proteomic studies clarified canonical synaptic proteins that are common to many types of synapses. However, proteins of diversified functions in a subset of synapses are largely hidden because of their low abundance or structural similarities to abundant proteins. To overcome this limitation, we have developed an “ultra-definition” (UD) subcellular proteomic workflow. Using purified synaptic vesicle (SV) fraction from rat brain, we identified 1,466 proteins, three times more than reported previously. This refined proteome includes all canonical SV proteins, as well as numerous proteins of low abundance, many of which were hitherto undetected. Comparison of UD quantifications between SV and synaptosomal fractions has enabled us to distinguish SV-resident proteins from potential SV-visitor proteins. We found 134 SV residents, of which 86 are present in an average copy number per SV of less than one, including vesicular transporters of nonubiquitous neurotransmitters in the brain. We provide a fully annotated resource of all categorized SV-resident and potential SV-visitor proteins, which can be utilized to drive novel functional studies, as we characterized here Aak1 as a regulator of synaptic transmission. Moreover, proteins in the SV fraction are associated with more than 200 distinct brain diseases. Remarkably, a majority of these proteins was found in the low-abundance proteome range, highlighting its pathological significance. Our deep SV proteome will provide a fundamental resource for a variety of future investigations on the function of synapses in health and disease.

The functions of eukaryotic cells, in all their complexity, depend upon highly specific compartmentalization into subcellular domains, including organelles. These compartments represent functional units characterized by specific supramolecular protein complexes. A major goal of modern biology is to establish an exhaustive, quantitative inventory of the protein components of each intracellular compartment. Such inventories are points of departure, not only for functional understanding and reconstruction of biological systems, but also for a multitude of investigations, such as evolutionary diversification and derivation of general principles of biological regulation and homeostasis.Essential to communication within the nervous system, chemical synapses constitute highly specific compartments that are connected by axons to frequently distant neuronal cell bodies. Common to all chemical synapses are protein machineries that orchestrate exocytosis of synaptic vesicles (SVs) filled with neurotransmitters in response to presynaptic action potentials (APs), resulting in activation of postsynaptic receptors. Moreover, synapses are composed of structurally and functionally distinct subcompartments, such as free and docked SVs, endosomes, active zones (AZs) at the presynaptic side, and receptor-containing membranes with associated scaffold proteins on the postsynaptic side. Thus, it is not surprising that mass spectrometry (MS)-based proteomics, combined with subcellular fractionation, yields protein inventories of high complexity. For instance, >2,000 protein species were identified in synaptosomes (1), ∼400 in the SV fraction (2), ∼1,500 in postsynaptic densities (3), and ∼100 in an AZ-enriched preparation (4).While these studies provide insights into the protein composition of synaptic structures, they are still inherently limited for two reasons. First, synapses are functionally diverse with respect to the chemical nature of their neurotransmitters, as well as their synaptic strength, kinetics, and plasticity properties (5). Therefore, analyzed subcellular fractions represent “averages” of a great diversity of synapses (6) or SVs (2). The second limitation is that proteins known to be present in specific subsets were not found in these studies, despite the unprecedented sensitivity of modern mass spectrometers. In fact, many functionally critical synaptic proteins have remained undetected. For example, the synaptotagmin (Syt) family, major Ca²⁺ sensors of SV exocytosis, comprises >15 members, of which only 5 had been identified in previous SV proteomics (2, 4, 7). Missing isoforms included Syt7, involved in asynchronous transmitter release (8), synaptic plasticity (9), and SV recycling (10). Likewise, the vesicular transporters for monoamines (VMATs) and acetylcholine (VAChT) neurotransmitters were missing in these studies. Clearly, known components of the diversified synaptic proteome have been missing, and it is not possible to predict how many more such proteins remain hidden.What are the reasons for the continuing incompleteness of the synaptic protein inventory? Proteome identification and quantification rely heavily on MS detectability of peptides generated by digestion of extracted proteins with sequence-specific enzymes, such as trypsin. However, in MS analysis of complex biological samples, peptide signals from a few abundant proteins often mask those that are less abundant. Additionally, the probability of obtaining peptides with similar masses, but different amino acid sequences, increases with increasing sample complexity (11, 12). To overcome these limitations, we have elaborated a workflow with dual-enzymatic protein digestion in sequence combined with an extensive peptide separation prior to MS analysis. As proof of concept, we have utilized purified SV fractions from rat whole brain, which serve as a benchmark for quantitative organellar proteomics (2). As a result, we detected ∼1,500 proteins in the SV fraction, three times more than reported previously. This proteome not only covers all known canonical SV proteins but also contains proteins previously overlooked, such as the low-abundance Syts and SV transporters. Moreover, peptide quantification allowed for differentiating “SV-resident” from “SV-visitor” proteins. In fact, most “SV-resident” proteins revealed in our SV proteomics are of low abundance, with an average copy number of less than 1 per SV, suggesting a larger molecular and functional diversity of SVs than previously thought. Remarkably, more than 200 proteins detected in the SV fraction are genetically associated with brain disorders, 76% of which were previously hidden.