Novel biochemical, structural, and systems insights into inflammatory signaling revealed by contextual interaction proteomics.

Protein-protein interactions (PPIs) represent the main mode of the proteome organization in the cell. In the last decade, several large-scale representations of PPI networks have captured generic aspects of the functional organization of network components but mostly lack the context of cellular states. However, the generation of context-dependent PPI networks is essential for structural and systems-level modeling of biological processes-a goal that remains an unsolved challenge. Here we describe an experimental/computational strategy to achieve a modeling of PPIs that considers contextual information. This strategy defines the composition, stoichiometry, temporal organization, and cellular requirements for the formation of target assemblies. We used this approach to generate an integrated model of the formation principles and architecture of a large signalosome, the TNF-receptor signaling complex (TNF-RSC). Overall, we show that the integration of systems- and structure-level information provides a generic, largely unexplored link between the modular proteome and cellular function.

Global profiling of protein complex dynamics with an experimental library of protein interaction markers.

Methods to systematically monitor protein complex dynamics are needed. We introduce serial ultrafiltration combined with limited proteolysis-coupled mass spectrometry (FLiP-MS), a structural proteomics workflow that generates a library of peptide markers specific to changes in PPIs by probing differences in protease susceptibility between complex-bound and monomeric forms of proteins. The library includes markers mapping to protein-binding interfaces and markers reporting on structural changes that accompany PPI changes. Integrating the marker library with LiP-MS data allows for global profiling of protein-protein interactions (PPIs) from unfractionated lysates. We apply FLiP-MS to Saccharomyces cerevisiae and probe changes in protein complex dynamics after DNA replication stress, identifying links between Spt-Ada-Gcn5 acetyltransferase activity and the assembly state of several complexes. FLiP-MS enables protein complex dynamics to be probed on any perturbation, proteome-wide, at high throughput, with peptide-level structural resolution and informing on occupancy of binding interfaces, thus providing both global and molecular views of a system under study.