Global Proteomic Profiling of Drosophila Ovary: A High-resolution, Unbiased, Accurate and Multifaceted Analysis

Background: Drosophila melanogaster ovary serves as an attractive model system for the investigation of the cell cycle, death, signaling, migration, differentiation, development and stemness. By employing the 3750/+ heterozygote fly strain that carries specific functions in the follicle cell compartment, and a reliable control in GAL4/UAS-based transgenic technology, we herein characterized the protein-expression profiling of D. melanogaster ovary by applying high-resolution proteomic tools and bioinformatics programs. Materials and Methods: Whole-cell total protein extracts derived from 3750/+ fly ovaries were prepared under highly denaturing conditions and after tryptic digestion, their cognate peptides were processed to liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) analysis in a high-resolution LTQ Orbitrap Elite instrument. Obtained protein data were analyzed through use of UniProt, DAVID, KEGG and PANTHER bioinformatics platforms. Results: The 7,583 unique peptides identified show that fly ovary contains at least 2,103 single proteins, which are distributed to all egg chamber compartments, in cytoplasm, membrane and nucleus, compartmentalized into major cellular organelles, and categorized into critical macromolecular assemblies. Among the recognized specific functions, nucleic acid binding, hydrolase, oxidoreductase, transporter and vesicle- mediated trafficking activities were the most prevalent. Determinants implicated in cellular metabolism and gene expression are represented by ~41% and ~17% of the ovarian proteome, respectively. Surprisingly, several proteins were found engaged in aging, immune response and neurogenesis. All major signaling pathways were detected, while apoptotic and non- apoptotic cell death programs were also identified. Remarkably, proteins involved in tumor formation, neurodegenerative and inflammatory diseases were also recognized. The successful remodeling of the proteasome and nearly complete molecular reconstruction of the citrate cycle and fatty acid degradation pathways demonstrate the efficacy, accuracy and fidelity of our combined proteomics/bioinformatics approach. Conclusion: Global proteomic characterization of D. melanogaster ovary allows the discovery of novel regulators and pathways, and provides a systemic view of networks that govern ovarian pathophysiology and embryonic development in fly species as well in humans. Drosophila melanogaster has been established as a versatile and dynamic model system for investigating several aspects of cell and developmental biology, mainly due to its short- life cycle and powerful genetics. The high level of structural