The evolution of reproductive barriers is fundamental to the formation of new species and can help us understand the diversification of life on Earth. These reproductive barriers often take the form of hybrid incompatibilities, where genes derived from two different species no longer interact properly. Theory predicts that incompatibilities involving multiple genes should be common and that rapidly evolving genes will be more likely to cause incompatibilities, but empirical evidence has lagged behind these predictions. Here, we describe a mitonuclear incompatibility involving three genes within respiratory Complex I in naturally hybridizing swordtail fish. Individuals with specific mismatched protein combinations fail to complete embryonic development while those heterozygous for the incompatibility have reduced function of Complex I and unbalanced representation of parental alleles in the mitochondrial proteome. We localize the protein-protein interactions that underlie the incompatibility and document accelerated evolution and introgression in the genes involved. This work thus provides a precise characterization of the genetic architecture, physiological impacts, and evolutionary origin of a multi-gene incompatibility impacting naturally hybridizing species.

The analytical scale of most mass-spectrometry-based targeted proteomics assays is usually limited by assay performance and instrument utilization. A recently introduced method, called triggered by offset, multiplexed, accurate mass, high resolution, and absolute quantitation (TOMAHAQ), combines both peptide and sample multiplexing to simultaneously improve analytical scale and quantitative performance. In the present work, critical technical requirements and data analysis considerations for successful implementation of the TOMAHAQ technique based on the study of a total of 185 target peptides across over 200 clinical plasma samples are discussed. Importantly, it is observed that significant interference originate from the TMTzero reporter ion used for the synthetic trigger peptides. This interference is not expected because only TMT10plex reporter ions from the target peptides should be observed under typical TOMAHAQ conditions. In order to unlock the great promise of the technique for high throughput quantification, here a post-acquisition data correction strategy to deconvolute the reporter ion superposition and recover reliable data is proposed.