Label-free proteomic comparison reveals ciliary and nonciliary phenotypes of IFT-A mutants.

DIFFRAC is a powerful method for systematically comparing proteome content and organization between samples in a high-throughput manner. By subjecting control and experimental protein extracts to native chromatography and quantifying the contents of each fraction using mass spectrometry, it enables the quantitative detection of alterations to protein complexes and abundances. Here, we applied DIFFRAC to investigate the consequences of genetic loss of Ift122, a subunit of the intraflagellar transport-A (IFT-A) protein complex that plays a vital role in the formation and function of cilia and flagella, on the proteome of Tetrahymena thermophila. A single DIFFRAC experiment was sufficient to detect changes in protein behavior that mirrored known effects of IFT-A loss and revealed new biology. We uncovered several novel IFT-A-regulated proteins, which we validated through live imaging in Xenopus multiciliated cells, shedding new light on both the ciliary and non-ciliary functions of IFT-A. Our findings underscore the robustness of DIFFRAC for revealing proteomic changes in response to genetic or biochemical perturbation.

Ancient eukaryotic protein interactions illuminate modern genetic traits and disorders.

All eukaryotes share a common ancestor from roughly 1.5 - 1.8 billion years ago, a single-celled, swimming microbe known as LECA, the Last Eukaryotic Common Ancestor. Nearly half of the genes in modern eukaryotes were present in LECA, and many current genetic diseases and traits stem from these ancient molecular systems. To better understand these systems, we compared genes across modern organisms and identified a core set of 10,092 shared protein-coding gene families likely present in LECA, a quarter of which are uncharacterized. We then integrated >26,000 mass spectrometry proteomics analyses from 31 species to infer how these proteins interact in higher-order complexes. The resulting interactome describes the biochemical organization of LECA, revealing both known and new assemblies. We analyzed these ancient protein interactions to find new human gene-disease relationships for bone density and congenital birth defects, demonstrating the value of ancestral protein interactions for guiding functional genetics today.