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The synapse is the most characteristic feature of the brain that allows the flow of information encoding our cognitive functions, behavior and memory. Slight perturbations in synaptic function can derive in wide range of psychiatric, neurodevelopmental and neurodegenerative disorders. The aim of this thesis was to investigate the synaptic proteome and interactome in order to gain insights in the molecular mechanisms underlying synaptic function. To this end, we exploited the potential of multiple advanced mass spectrometry methodologies for protein identification, quantification, and protein interaction determination. In chapter 2, I investigated the molecular development of the synapse. This process requires prominent changes of the synaptic proteome and potentially involves thousands of different proteins at every synapse. We analyzed the cortical synaptic membrane proteome of juvenile, adolescent and adult mice brains using iTRAQ-based DDA quantitative proteomics. In several cases, proteins from a single functional molecular entity, e.g., subunits of the NMDA receptor, showed differences in their temporal regulation, which may reflect specific synaptic development features of connectivity, strength and plasticity. We also evaluated the function of Cxadr, a protein with high expression level at early stages and a fast decline in expression during neuronal development. Knockdown of the expression of Cxadr in cultured primary mouse neurons revealed a significant decrease in synapse density. Altogether, these results reveal the expression profile of synaptic proteome during development and provide new insights into the molecular processes underlying synaptogenesis and synapse maturation. In chapter 3, I explored the mechanism behind the synaptic modulation mediated by the metabotropic glutamate receptor 5. mGluR5 plays a major role in the modulation of synaptic function and plasticity, as well as in several brain disorders. Despite robust pre-clinical data, mGluR5 antagonists failed in several clinical trials, highlighting the need for a better understanding of the mechanisms underlying mGluR5 function. Using a proteomic approach, we determined the molecular response of the synapse to a reduction of mGluR5 activity by pharmacological inhibition and gene deletion. In both cases, the most prominent response of the synaptic proteome was the change in protein expression of key mitochondrial pathways. Together with this, we observed morphological and functional alterations of mitochondria in mGluR5 KO synapses. Our findings provide new insight into a functional connection of mGluR5 and specific mitochondrial function. In chapter 4, I applied XL-MS as entry into the synapse interactome, in particular to reveal the architecture and assembly of synaptic protein complexes. As a result, we generated to the first large-scale cross-linking repository in the brain. The reliability of the data was validated by several approaches as we deemed necessary for a recent methodology. In addition, a large part of the crosslink data contains novel information which allowed us to identify novel protein partners, to model protein conformational dynamics, and to delineate within and between protein interactions of main synaptic constituents, such as Camk2, the AMPA-type glutamate receptor, and associated proteins. Given the molecular complexity of the synapse and the large amount and depth of the data generated, we provided the complete dataset as an interactive web-based platform for further investigations (http://xlink.cncr.nl). Together, we generated one of the largest cross-linking collections that provided new entries into exploration of protein structures and interactions. Collectively, the application and development of multiple proteomic methodologies allowed us to reveal several aspects of the molecular architecture of the synapse, including protein composition, function, structure and interaction. Beyond the new insights uncovered for specific proteins in this thesis, the data resources generated can be further used for probing additional proteins and contributes to improve our understanding of synapse function and brain disease.