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We describe a next-generation Drosophila protein interaction map-"DPIM2"-established from affinity purification-mass spectrometry of 5,805 baits, covering the largest fraction of the Drosophila proteome. The network contains 32,668 interactions among 3,644 proteins, organized into 632 clusters representing putative functional modules. Our analysis expands the pool of known protein interactions in Drosophila, provides annotation for poorly studied genes, and postulates previously undescribed protein interaction relationships. The predictive power and functional relevance of this network are probed through the lens of the Notch signaling pathway, and we find that newly identified members of complexes that include known Notch modifiers can also modulate Notch signaling. DPIM2 allows direct comparisons with a recently published human protein interaction network, defining the existence of functional interactions conserved across species. Thus, DPIM2 defines a valuable resource for predicting protein co-complex memberships and functional associations as well as generates functional hypotheses regarding specific protein interactions., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Thousands of interactions assemble proteins into modules that impart spatial and functional organization to the cellular proteome. Through affinity-purification mass spectrometry, we have created two proteome-scale, cell-line-specific interaction networks. The first, BioPlex 3.0, results from affinity purification of 10,128 human proteins-half the proteome-in 293T cells and includes 118,162 interactions among 14,586 proteins. The second results from 5,522 immunoprecipitations in HCT116 cells. These networks model the interactome whose structure encodes protein function, localization, and complex membership. Comparison across cell lines validates thousands of interactions and reveals extensive customization. Whereas shared interactions reside in core complexes and involve essential proteins, cell-specific interactions link these complexes, "rewiring" subnetworks within each cell's interactome. Interactions covary among proteins of shared function as the proteome remodels to produce each cell's phenotype. Viewable interactively online through BioPlexExplorer, these networks define principles of proteome organization and enable unknown protein characterization., Competing Interests: Declaration of interests J.W.H. is a founder and scientific advisory board member of Caraway Therapeutics and a Founding Scientific Advisor for Interline Therapeutics., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The physiology of a cell can be viewed as the product of thousands of proteins acting in concert to shape the cellular response. Coordination is achieved in part through networks of protein-protein interactions that assemble functionally related proteins into complexes, organelles, and signal transduction pathways. Understanding the architecture of the human proteome has the potential to inform cellular, structural, and evolutionary mechanisms and is critical to elucidating how genome variation contributes to disease. Here we present BioPlex 2.0 (Biophysical Interactions of ORFeome-derived complexes), which uses robust affinity purification-mass spectrometry methodology to elucidate protein interaction networks and co-complexes nucleated by more than 25% of protein-coding genes from the human genome, and constitutes, to our knowledge, the largest such network so far. With more than 56,000 candidate interactions, BioPlex 2.0 contains more than 29,000 previously unknown co-associations and provides functional insights into hundreds of poorly characterized proteins while enhancing network-based analyses of domain associations, subcellular localization, and co-complex formation. Unsupervised Markov clustering of interacting proteins identified more than 1,300 protein communities representing diverse cellular activities. Genes essential for cell fitness are enriched within 53 communities representing central cellular functions. Moreover, we identified 442 communities associated with more than 2,000 disease annotations, placing numerous candidate disease genes into a cellular framework. BioPlex 2.0 exceeds previous experimentally derived interaction networks in depth and breadth, and will be a valuable resource for exploring the biology of incompletely characterized proteins and for elucidating larger-scale patterns of proteome organization.