A mechanistic understanding of a living cell requires the characterization of the architecture and dynamic assembly of its macromolecular complexes. The proposed project describes a novel integrative approach to determine the topology of protein networks and higher order assemblies using chemical crosslinking, state of the art mass spectrometry (MS) and cryo-electron tomography (cryo-ET). This MS based strategy will enable the high-throughput detection and integration of structural data of biological systems. As a promising target we have chosen a giant key cell division structure, the human kinetochore, which is crucial for faithful chromosome segregation during cell division. Up to now, a comprehensive understanding of its cell cycle-regulated assembly and the nature of interactions between its protein components remain elusive. To investigate the architecture of the kinetochore we propose to apply a novel MS based crosslinking strategy that allows monitoring of protein interfaces at primary structure level. We plan to combine this technology with cryo-ET of the native kinetochore structure and of affinity-purified kinetochore complexes. Based on the spatial restraints obtained by MS, subunits can be fitted into electron density maps with high confidence, which will facilitate the generation of a 3D topological map of the entire kinetochore. Currently, there is no mechanistic understanding of the temporal dynamics of kinetochore protein assembly/disassembly which presumably involves cell cycle-controlled synthesis and degradation, as well as post-translational cues. To measure alterations in protein levels and phosphorylation status, we will investigate purified subcomplexes or whole cell lysates by quantitative targeted MS methods pioneered by the host institution. The resulting abundance information of proteins and their phosphorylation sites promises to reveal the stoichiometric relationships of kinetochore components and their cell cycle-regulated assembly.