Comparison of the secretome of follicle cells during developmental phagoptosis and starvation-induced apoptotic death of nurse cells in Drosophila melanogaster oogenesis.

Associated preprint - https://www.biorxiv.org/content/10.1101/2024.03.12.584558v1

Keywords 

secretome, Drosophila, oogenesis, cell death, HRP-KDEL

Sample Processing Protocol

pDisplay-ss-V5-HRPKDEL was obtained from Dr. Alice Ting (Stanford).The insert was PCR-amplified and cloned into the pENTR/D-TOPO cloning kit (Invitrogen) and pTW vector (Drosophila Genome Resource Center, RRID:DGRC_1129)) using Gateway cloning (Invitrogen). The plasmid was purified by Qiagen Midiprep kit, confirmed by sequencing and sent to BestGene (Chino Hills, CA) for injection into Drosophila embryos. Freshly dissected ovaries were incubated in 300 μL of 500 uM biotin phenol for 30 minutes at room temperature rotating. Samples were then rinsed with 1X PBS twice and the biotinylation reaction was initiated by adding 1 mM H2O2 in PBS to the samples for 1 minute and rotating at room temperature. Ovaries were quickly washed with quencher solution (10 mM sodium ascorbate, 5 mM Trolox (Sigma-Aldrich), 10 mM sodium azide, then lysed in 100 μL RIPA buffer with quencher solution for 5 min on ice. RIPA buffer was composed of: 50 μL 1M Tris-HCl, 150 μL 5M NaCl, 50 μL of 10% SDS, 250 μL of 10% Sodium Deoxycholate, 500 μL of 10% TritonX-100, 50 μL of 100X Protease Inhibitor (Sigma-Aldrich – P8849), 50 μL of 100 mM PMSF, 3.550 mL of diH20. Tissue was homogenized by motorized pestle and centrifuged at 16.1g for 10 min at 4°C. Clarified sample (clear middle layer) was transferred to a new tube and snap frozen in liquid nitrogen. Biotinylated proteins were pulled down using streptavidin magnetic beads. Beads from biotinylated protein pull-down were washed with 100 mM triethylammonium bicarbonate. Peptides were eluted from beads by on-bead trypsin digestion with 1μg Trypsin (Pierce) in 100 mM triethylammonium bicarbonate overnight rotating at 37°C. Peptides were desalted using C18 ZipTip (Millipore) and subjected liquid chromatography coupled to tandem mass spectrometry on a Q Exactive HF-X (Thermo Fisher Scientific). Data-dependent fragmentation used collision-induced dissociation.

Data Processing Protocol

RAW files were searched using MaxQuant under standard settings using the UniProt Drosophila melanogaster database, allowing for two missed trypsin cleavage sites, variable modifications for N-terminal acetylation, and methionine oxidation. Candidate peptides and protein identifications were filtered on the basis of a 1% false discovery rate.

Experiment Type

Data-dependent acquisition

Files included

MaxQuant search files - tables.pdfallPeptides.txtevidence-mss.txtevidence.txtGlyGly_KSites.txtHRPKDEL_dmel.xlsxlibraryMatch.txtmatchedFeatures.txtmodificationSpecificPeptides.txtms3Scans.txtmsms.txtmsmsScans.txtmsScans.txtmzRange.txtOxidation_MSites.txtparameters.txtpeptides.txtPhospho_STYSites.txtproteinGroups.txtQQTGG_KSites.txtsummary.txt