The old and the new: discovery proteomics identifies putative novel seminal fluid proteins in Drosophila.

Seminal fluid proteins (SFPs), the non-sperm component of male ejaculates produced by male accessory glands, are viewed as central mediators of reproductive fitness. SFPs effect both male and female post-mating functions and show molecular signatures of rapid adaptive evolution. While Drosophila melanogaster is the dominant insect model for understanding SFP evolution, understanding of SFP evolutionary causes and consequences require additional comparative analyses of close and distantly-related taxa. While SFP identification was historically challenging, advances in label-free quantitative proteomics expands the scope of studying other systems to further advance the field. Focused studies of SFPs has so far overlooked the proteomes of male reproductive glands and their inherent complex protein-protein networks for which there is little information on the overall signals of molecular evolution. Here we applied label-free quantitative proteomics to identify the accessory gland proteome and secretome in Drosophila pseudoobscura, a close relative of D. melanogaster, and use the dataset to identify both known and putative novel SFPs. Using this approach we identified 163 putative SFPs, 32% of which overlapped with previously identified D. melanogaster SFPs and show that SFPs with known extracellular annotation evolve more rapidly than other proteins produced by or contained within the accessory gland. Our results will further the understanding of the evolution of SFPs and the underlying male accessory gland proteins that mediate reproductive fitness of the sexes.