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Single-Organ Proteomics in Drosophila melanogaster

Abstract #167 Posted on26 March 202512 May 2025 12 May 2025 13h03

Abstract

The combination of genetic accessibility, organ complexity, evolutionary conservation, and cost-efficiency makes Drosophila melanogaster (Dm) an exceptional model system for biomedical and fundamental biological research. Hereby we present a proteomic pipeline to, for the first time, comprehensively map single-organ proteomes in Dm.

Larvae were manually dissected to isolate individual organs, including the brain, ring gland, eye disc, leg disc, haltere disc, wing disc, salivary gland, fat body, testis, and gastric caeca/proventriculus/garland cells. Each single-organ was deposited into low-binding tubes containing an extraction buffer based on the protocol described by Ye et al. (2024). Specific protein extraction approach was performed combing freeze and thaw cycles with double protease digestion (LysC + Trypsin). Acquisition was performed in an EVOSEP (whisper method) on-line coupled to a Tims tof Pro by acquiring in DIA mode. Searches were performed with Dia-NN.

The number of proteins identified per organ ranged from 2,293 (ring gland) to 5,937 (brain) in at least three out of four replicates. In total, the combined dataset across all organs comprised over 8,000 identified proteins.

As anticipated, principal component analysis (PCA) revealed clear separation between the proteomes of most organs, confirming distinct protein expression profiles. These findings demonstrate the feasibility of high-resolution proteomic characterization at the level of individual organs in D

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Larvae were manually dissected to isolate individual organs, including the brain, ring gland, eye disc, leg disc, haltere disc, wing disc, salivary gland, fat body, testis, and gastric caeca/proventriculus/garland cells. Each single-organ was deposited into low-binding tubes containing an extraction buffer based on the protocol described by Ye et al. (2024). Specific protein extraction approach was performed combing freeze and thaw cycles with double protease digestion (LysC + Trypsin). Acquisition was performed in an EVOSEP (whisper method) on-line coupled to a Tims tof Pro by acquiring in DIA mode. Searches were performed with Dia-NN.

The number of proteins identified per organ ranged from 2,293 (ring gland) to 5,937 (brain) in at least three out of four replicates. In total, the combined dataset across all organs comprised over 8,000 identified proteins.

As anticipated, principal component analysis (PCA) revealed clear separation between the proteomes of most organs, confirming distinct protein expression profiles. These findings demonstrate the feasibility of high-resolution proteomic characterization at the level of individual organs in D

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