Comparative Clustering (CompaCt) of eukaryote complexomes identifies novel interactions and sheds light on protein complex evolution

Complexome profiling allows large-scale, untargeted, and comprehensive characterization of protein complexes in a biological sample using a combined approach of separating intact protein complexes e.g., by native gel electrophoresis, followed by mass spectrometric analysis of the proteins in the resulting fractions. Over the last decade, its application has resulted in a large collection of complexome profiling datasets. While computational methods have been developed for the analysis of individual datasets, methods for large-scale comparative analysis of complexomes from multiple species are lacking. Here, we present Comparative Clustering (CompaCt), that performs fully automated integrative analysis of complexome profiling data from multiple species, enabling systematic characterization and comparison of complexomes. CompaCt implements a novel method for leveraging orthology in comparative analysis to allow systematic identification of conserved as well as taxon-specific elements of the analyzed complexomes. We applied this method to a collection of 53 complexome profiles spanning the major branches of the eukaryotes. We demonstrate the ability of CompaCt to robustly identify the composition of protein complexes, and show that integrated analysis of multiple datasets improves characterization of complexes from specific complexome profiles when compared to separate analysis. We identified novel candidate interactors and complexes in a number of species from previously analyzed datasets, like the emp24, the V-ATPase and mitochondrial ATP synthase complexes. Lastly, we demonstrate the utility of CompaCt for the automated large-scale characterization of the complexome of the mosquito Anopheles stephensi shedding light on the evolution of metazoan protein complexes. CompaCt is available from . Author summary Proteins carry out essential functions in the majority of processes in life, often by binding with other proteins to form multiprotein complexes. State of the art experimental techniques such as complexome profiling enable large-scale identification of protein complexes in a biological sample. With the increase in use of this method in recent years these experiments have been performed on a variety of species, of which the results are publicly available. Combining the results from these experiments presents a computational challenge, but could identify novel protein complexes and provide insights into their evolution. Here, we introduce CompaCt as a method to integrate complexome profiles from multiple species enabling automatic large-scale characterization of protein complexes. It identifies commonalities as well as the differences between species. By applying CompaCt to a collection of complexome profiles, we identified candidate complexes and interacting proteins in a number of species that were not detected in previous separate analyses of these datasets. In doing so we shed light on the evolutionary origin of several protein complex members, pinpointed the function of biomedically relevant proteins, whose role was previously unknown, and performed the first investigation of the Anopheles stephensi complexome, a mosquito that transmits the malaria parasite. ### Competing Interest Statement The authors have declared no competing interest.