Glomerular filtration is a vital physiological process removing waste products from the circulation and this process occurs across the glomerular filtration barrier (GFB). The cells and extracellular matrix (ECM), which form this barrier, are exposed to forces during ultrafiltration and special adaptation is required to withstand these forces. Dysfunction in cellular adhesion machinery or ECM assembly within the GFB causes loss of selective glomerular filtration, however, the mechanisms governing these processes are poorly understood. To this end we sought to characterise the glomerular ECM and adhesion machinery using high throughput mass spectrometry (MS)-based proteomics.MS of human glomerular ECM identified a highly complex extracellular niche, revealing the potential involvement of novel ECM proteins in glomerular development and disease processes. Furthermore we identified that glomerular cells in culture had distinct ECM proteomes and interestingly, coculture experiments demonstrated that the ECM proteome was influenced by cellular crosstalk and had a closer resemblance to glomerular ECM in vivo. Protein network analyses of in vivo and in vitro ECM datasets revealed a common core of highly connected structural ECM proteins that may be important for glomerular ECM assembly. To understand how this ECM proteome altered in disease, we studied mice with mild glomerular dysfunction. Here, transcriptomic and proteomic analyses identified alterations in ECM composition and 3D electron microscopy revealed striking ultrastructural changes in glomerular ECM. MS-based proteomics was next applied to the analysis of glomerular podocyte adhesion complexes, leading to the discovery that the actin cytoskeletal regulators and trafficking machinery are recruited to adhesions sites in an ECM-ligand dependent manner. Furthermore, these differences functionally altered cell shape and adhesion strength. These same analyses were applied to podocyte cell-cell junctions, revealing an unexpected overlap of cell-ECM and cell-cell adhesion machinery.Overall, these findings demonstrate for the first time the complexity of the glomerular ECM and adhesion signalling complexes and reinforce the benefits of global, unbiased experimental approaches. In addition the results suggest that glomerular ECM composition, organisation and adhesion signalling are context dependent, and therefore, represent potential therapeutic targets.