PROTEOMIC ANALYSIS OF DROSOPHILA SEMINAL FLUID

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 除了精子，雄性果蝇在交配时还会向雌性传递数十种蛋白质。其中许多蛋白质影响雌性果蝇的生理和行为，其中一些已知在不同果蝇物种之间进化迅速。以前的计算工作已经预测了精液中转移的一组蛋白质，但这些预测只对少数蛋白质有效。在这里，我们提出了一种基于质谱学的方法来全面鉴定男性精液蛋白质组。果蝇将通过摄食标记有重氮同位素15-N的酵母来进行同位素标记。有标记的雄性将与未标记的雌性交配。交配后，我们将立即解剖雌性生殖道，并用质谱仪分析其中的蛋白质。雄性蛋白质将通过同位素标记与雌性蛋白质区分开来。通过这种方式，我们的目标是鉴定所有在果蝇精液中转移的蛋白质。如果这项初步实验成功，我们将继续用同样的方法测试不同品系的黑腹果蝇、面临不同交配条件的雄性果蝇以及其他果蝇种雄性转移的蛋白质水平和/或类型的差异。我们的总体目标是更好地了解交配过程中转移的蛋白质的功能和进化。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. In addition to sperm, Drosophila males transfer dozens of proteins to females upon mating. Many of these proteins affect the physiology and behavior of females, and several are known to evolve rapidly between different Drosophila species. Previous computational work has predicted the set of proteins transferred in seminal fluid, but these predictions have been validated for only a handful of proteins. Here, we propose a mass spectrometry-based approach to comprehensively identify the set of male seminal fluid proteins. Drosophila melanogaster flies will be isotopically labeled by feeding on yeast labeled with a heavy isotope of nitrogen, 15-N. Labeled males will be mated to unlabeled females. Immediately following mating, we will dissect the female reproductive tracts and analyze the proteins therein by mass spectrometry. Male proteins will be distinguished from female proteins by the isotopic label. In this way, we aim to identify all of the proteins transferred in Drosophila seminal fluid. If this preliminary experiment is successful, we will proceed to test by the same method for variation in the levels and/or types of proteins transferred by different strains of D. melanogaster, by males facing different mating conditions, and by males of other Drosophila species. Our overall goal is to better understand the functions and evolution of proteins transferred during mating.