Hidden proteome of synaptic vesicles in the mammalian brain Zacharie Taoufiqa,1, Momchil Ninovb, Alejandro Villar-Brionesc, Han-Ying Wanga, Toshio Sasakid, Michael C. Royc, Francois Beauchaina, Yasunori Morie, Tomofumi Yoshidae, Shigeo Takamorie, Reinhard Jahnb,1, and Tomoyuki Takahashia,1 aCellular and Molecular Synaptic Function Unit, Okinawa Institute of Science and Technology Graduate University, Okinawa 904-0495, Japan; bDepartment of Neurobiology, Max Planck Institute for Biophysical Chemistry, D-37077 Göttingen, Germany; cInstrumental Analysis Section, Okinawa Institute of Science and Technology Graduate University, Okinawa 904-0495, Japan; dImaging Section, Okinawa Institute of Science and Technology Graduate University, Okinawa 904-0495, Japan; and eLaboratory of Neural Membrane Biology, Graduate School of Brain Science, Doshisha University, 610-0394 Kyoto, Japan Contributed by Reinhard Jahn, November 11, 2020 (sent for review June 15, 2020; reviewed by Yukiko Goda, Stefan Hallermann, and August B. Smit) Current proteomic studies clarified canonical synaptic proteins that proteomics, combined with subcellular fractionation, yields pro- are common to many types of synapses. However, proteins of tein inventories of high complexity. For instance, >2,000 protein diversified functions in a subset of synapses are largely hidden species were identified in synaptosomes (1), ∼400 in the SV fraction because of their low abundance or structural similarities to abun- (2), ∼1,500 in postsynaptic densities (3), and ∼100 in an AZ- dant proteins. To overcome this limitation, we have developed an enriched preparation (4). “ultra-definition” (UD) subcellular proteomic workflow. Using pu- While these studies provide insights into the protein compo- rified synaptic vesicle (SV) fraction from rat brain, we identified sition of synaptic structures, they are still inherently limited for 1,466 proteins, three times more than reported previously. This two reasons. First, synapses are functionally diverse with respect refined proteome includes all canonical SV proteins, as well as nu- to the chemical nature of their neurotransmitters, as well as their merous proteins of low abundance, many of which were hitherto synaptic strength, kinetics, and plasticity properties (5). There- undetected. Comparison of UD quantifications between SV and syn- fore, analyzed subcellular fractions represent “averages” of a aptosomal fractions has enabled us to distinguish SV-resident pro- great diversity of synapses (6) or SVs (2). The second limitation teins from potential SV-visitor proteins. We found 134 SV residents, is that proteins known to be present in specific subsets were not NEUROSCIENCE of which 86 are present in an average copy number per SV of found in these studies, despite the unprecedented sensitivity of less than one, including vesicular transporters of nonubiquitous modern mass spectrometers. In fact, many functionally critical neurotransmitters in the brain. We provide a fully annotated re- synaptic proteins have remained undetected. For example, the source of all categorized SV-resident and potential SV-visitor pro- synaptotagmin (Syt) family, major Ca2+ sensors of SV exocytosis, teins, which can be utilized to drive novel functional studies, as we > characterized here Aak1 as a regulator of synaptic transmission. comprises 15 members, of which only 5 had been identified in Moreover, proteins in the SV fraction are associated with more than previous SV proteomics (2, 4, 7). Missing isoforms included Syt7, 200 distinct brain diseases. Remarkably, a majority of these proteins involved in asynchronous transmitter release (8), synaptic plas- was found in the low-abundance proteome range, highlighting its ticity (9), and SV recycling (10). Likewise, the vesicular trans- pathological significance. Our deep SV proteome will provide a fun- porters for monoamines (VMATs) and acetylcholine (VAChT) damental resource for a variety of future investigations on the func- tion of synapses in health and disease. Significance synapse | deep proteomics | synaptic vesicles | brain disorders | Mammalian central synapses of diverse functions contribute to neurotransmission highly complex brain organization, but the molecular basis of syn- aptic diversity remains open. This is because current synapse pro- he functions of eukaryotic cells, in all their complexity, depend teomics are restricted to the “average” composition of abundant Tupon highly specific compartmentalization into subcellular synaptic proteins. Here, we demonstrate a subcellular proteomic domains, including organelles. These compartments represent workflow that can identify and quantify the deep proteome of functional units characterized by specific supramolecular protein synaptic vesicles, including previously missing proteins present in a complexes. A major goal of modern biology is to establish an ex- small percentage of central synapses. This synaptic vesicle proteome haustive, quantitative inventory of the protein components of each revealed many proteins of physiological and pathological relevance, intracellular compartment. Such inventories are points of depar- particularly in the low-abundance range, thus providing a resource ture, not only for functional understanding and reconstruction of for future investigations on diversified synaptic functions and biological systems, but also for a multitude of investigations, such neuronal dysfunctions. as evolutionary diversification and derivation of general principles Author contributions: Z.T. designed research; Z.T., M.N., A.V.-B., H.-Y.W., T.S., M.C.R., F.B., of biological regulation and homeostasis. Y.M., and T.Y. performed research; M.N. contributed new reagents/analytic tools; Z.T. and Essential to communication within the nervous system, chem- A.V.-B. analyzed data; Z.T., S.T., R.J., and T.T. wrote the paper; R.J. provided technical ical synapses constitute highly specific compartments that are support; and R.J. and T.T. provided supervision. connected by axons to frequently distant neuronal cell bodies. Reviewers: Y.G., RIKEN Center for Brain Science; S.H., Leipzig University; and A.B.S., Fac- Common to all chemical synapses are protein machineries that ulty of Science, Vrij Universiteit. orchestrate exocytosis of synaptic vesicles (SVs) filled with neu- Competing interest statement: R.J. (author) and A.B.S. (reviewer) are coauthors on a 2019 SynGO consortium article [F. Koopmans et al., Neuron 103, 217–234 (2019)]. rotransmitters in response to presynaptic action potentials (APs), This open access article is distributed under Creative Commons Attribution License 4.0 resulting in activation of postsynaptic receptors. Moreover, syn- (CC BY). apses are composed of structurally and functionally distinct sub- 1To whom correspondence may be addressed. Email: [email protected], rjahn@ compartments, such as free and docked SVs, endosomes, active gwdg.de, or [email protected]. zones (AZs) at the presynaptic side, and receptor-containing This article contains supporting information online at https://www.pnas.org/lookup/suppl/ membranes with associated scaffold proteins on the postsynaptic doi:10.1073/pnas.2011870117/-/DCSupplemental. side. Thus, it is not surprising that mass spectrometry (MS)-based www.pnas.org/cgi/doi/10.1073/pnas.2011870117 PNAS Latest Articles | 1of11 Downloaded by guest on September 27, 2021 neurotransmitters were missing in these studies. Clearly, known the resolving power of the UD method is based upon improved components of the diversified synaptic proteome have been missing, workflow prior to MS analysis (SI Appendix, Fig. S1). Note that and it is not possible to predict how many more such proteins each sample used for our MS analyses was checked by electron remain hidden. microscopy (EM) and electrophoresis. Typical synaptosomal What are the reasons for the continuing incompleteness of the profiles were observed in P2′ samples whereas uniform vesicle synaptic protein inventory? Proteome identification and quanti- structures of 40 to 50 nm in diameter predominated SV fractions fication rely heavily on MS detectability of peptides generated by (Fig. 1E). Proteins extracted from the P2′ and SV fractions digestion of extracted proteins with sequence-specific enzymes, showed distinct sodium dodecyl sulfate polyacrylamide gel such as trypsin. However, in MS analysis of complex biological electrophoresis (SDS/PAGE) profiles (Fig. 1F). samples, peptide signals from a few abundant proteins often mask those that are less abundant. Additionally, the probability of Improved Quantification Revealed the Synaptic Organization and obtaining peptides with similar masses, but different amino acid Diversity of the SV Proteome. In quantitative MS, protein abun- sequences, increases with increasing sample complexity (11, 12). To dance can be determined using intensity-based absolute quanti- overcome these limitations, we have elaborated a workflow with fication (iBAQ), a label-free approach in which the summed dual-enzymatic protein digestion in sequence combined with an intensities of all unique peptides of a protein are divided by the extensive peptide separation prior to MS analysis. As proof of total number of unique peptides detected. Thus, the increased concept, we have utilized purified SV fractions from rat whole peptide recovery achieved with the UD method is expected to brain, which serve as a benchmark for quantitative organellar improve the accuracy of protein quantification. To test this as- proteomics (2). As a result,