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Multi-modal comparison of molecular programs driving nurse cell death and clearance in Drosophila melanogaster oogenesis

ABSTRACT: Project_description: We adapted proximity labeling to identify proteins trafficked through the endoplasmic reticulum in Drosophila follicle cells. To identify these proteins, we biochemically tagged and enriched proteins in the ER by expressing a genetically tagged fusion construct carrying horseradish peroxidase (HRP) fused to the ER-retention signal KDEL, along with secretion signal IgK leader sequence (ss). We expressed ss-HRP-KDEL-V5 in follicle cells, where it localized to the ER. We exposed dissected ovaries to biotin-phenol substrate and a brief pulse of H2O2, which catalyzed the biotinylation of the proteins in the vicinity of HRP. We identified 50 differentially abundant proteins in follicle cells in different states. Sample_processing_protocol: Freshly dissected ovaries were incubated in 300 uL 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 uL RIPA buffer with quencher solution for 5 min on ice. RIPA buffer was composed of: 50 uL 1M Tris-HCl, 150 uL 5M NaCl, 50 uL of 10% SDS, 250 uL of 10% Sodium Deoxycholate, 500 uL of 10% TritonX-100, 50 uL of 100X Protease Inhibitor (Sigma-Aldrich - P8849), 50 uL 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 deg. C. Clarified sample (clear middle layer) was transferred to a new tube and snap frozen in liquid nitrogen. Frozen protein samples were thawed on ice. Meanwhile, 50 uL streptavidin magnetic beads (Pierce 88817) were washed with 1 mL of RIPA lysis buffer twice. The beads were subsequently incubated with 90 uL of protein lysate (about 550 ug of protein) and an additional 500 uL of RIPA buffer was added to facilitate rotation for 1 hour at room temperature. Beads were pelleted on a magnetic rack and the supernatant (flow-through) was collected on ice. After each wash, the magnetic beads were transferred into new tubes and washed twice with 1 mL RIPA buffer, once with 1 mL KCl, once with 1 mL 0.1 M Na2CO3, once with 1 mL 2 M Urea in 10 mM Tris-HCl (pH 8), and twice with 1 mL RIPA buffer. RIPA buffer was removed, and the beads snap frozen in liquid nitrogen and stored at -80 deg. C. Beads from biotinylated protein pull-down were washed with 100 mM triethylammonium bicarbonate. Peptides were eluted from beads by on-bead trypsin digestion with 1ug Trypsin (Pierce) in 100 mM triethylammonium bicarbonate overnight rotating at 37 deg. 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. Raw intensities from the MaxQuant spectral database search engine were imported into R for downstream analysis. QC metrics such as number of unique peptides identified, percent of contaminants and reverse decoys detected, and total sum of intensities at the replicate level and condition level were computed and visualized using artMS v1.12.0. Spurious hits such as reverse decoys and potential contaminants were removed. R package DEP v1.16.0 was used to remove proteins not identified in both replicates simultaneously and variance stabilizing transformation was applied to normalize the intensities. Missing values were imputed using the k-nearest neighbors imputation method, followed by differential abundance estimation and multiple hypothesis testing correction in limma, all of which were implemented in DEP.

INSTRUMENT(S): Q Exactive HF-X

ORGANISM(S): Drosophila Melanogaster (ncbitaxon:7227)

SUBMITTER: Kim McCall

PROVIDER: MSV000096618 | MassIVE | Mon Dec 09 08:02:00 GMT 2024

REPOSITORIES: MassIVE

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Project description:Female Sprague-Dawley rats were randomly separated into two groups: i) trained (Ex group) and ii) sedentary (Cont group). Animals from the Ex group were adapted to treadmill exercise for two consecutive weeks, involving a gradual increase in the running time till 60 min/day at 20 m/min, 0% grade, 5 days/week. This exercise program remained constant until the end of the 54 weeks. Mitochondria isolation from 10 animals (5 Ex group and 5 Cont group) was performed using the conventional methods of differential centrifugation. Mitochondrial protein extracts were reduced with dithiothreitol, alkylated with iodoacetamide and digested with trypsin using the FASP procedure (18). Briefly, each sample was solubilized in 4 % SDS, 0.1 M DTT, 0.1 M HEPES and incubated for 30 min at 60 C. Then, samples were mixed with 0.2 mL of a solution of 8 M Urea, 0.1 M HEPES, pH 8.5 (UH solution), loaded into the filtration devices (3k Microcon, Millipore), centrifuged at 14,000 g for 20 min, and washed twice with UH solution. After centrifugation, the concentrates were alkylated with 50 mM iodoacetamide in UH solution (dark, 25 C, 20 min), and washed twice with UH solution. The concentrate was diluted with 0.1 mL of 50 mM ammonium bicarbonate (ABC) and digested overnight (37 C) with the addition of 2 ug of trypsin diluted in 30 uL of 50 mM ABC. The resulting tryptic peptides were collected by centrifugation and the filter were washed twice with 50 uL of 50 mM ABC. Eluted peptides were acidified with 10 uL of formic acid and cleaned up on a homemade Empore C18 column (3M, St. Paul, MN, USA) (ref) for subsequent phospho-enrichment and/or analysis by LC-MS/MS. The dried protein digest was dissolved with 10 uL (3 % ACN, 0.1 % TFA), diluted 1:5 with loading buffer (1 M glycolic acid, 5 % TFA, 80 % ACN) and phosphopeptides were enriched on TiO2-beads as previously described (19). Briefly 5 mg of "Titansphere TiO2 5 um" were suspended in 100 uL of 100 % ACN, immobilized in a pipette-column and washed using 5 uL of loading buffer. Each sample was loaded in each pipette-column and then washed with 30 uL of washing buffer (1 % TFA, 80 % ACN). Phosphopeptides were eluted from the beads with 25 uL of 25 % ACN (v/v) containing 25 % NH4OH (m/v), acidified with 10 uL of 10 % TFA and vacuum- concentrated to approximately 4 uL. Samples were finally diluted to 10 uL with H2O + 0.1% formic acid prior to injection. The peptides mixtures were analyzed by online nanoflow liquid chromatography tandem mass spectrometry (nanoLC-MS/MS) on an EASY-nLC system (Proxeon Biosystems, Odense, Denmark) connected to the LTQ Orbitrap Velos instrument (Thermo Fisher Scientific, Bremen, Germany) through a nanoelectrospray ion source. Six microliters of the peptide mixture were auto-sampled directly onto the analytical HPLC column (120 mm x75 um I.D., 3 um particle size (Nikkyo Technos Co., Ltd.) with a 120-min gradient from 5 % to 50 % ACN in 0.1 % FA. The effluent from the HPLC column was directly electrosprayed into the mass spectrometer. The LTQ Orbitrap Velos instrument was operated in data-dependent acquisition mode to automatically switch between full scan MS and MS/MS acquisition. The MS survey scan was performed in the FT cell recording a window between 350 and 2000 m/z. The resolution was set to 60 000, and the automatic gain control was set to 1,000,000 ions with a maximal acquisition time of 400 ms. The 20 most intense peptide ions with charge states great than or equal to 2 were sequentially isolated to a target value of 5,000 and fragmented in the high-pressure linear ion trap by low-energy CID with normalized collision energy of 35% Q value of 0.25, and an activation time of 10 ms. Raw files were processed using Proteome Discoverer version 1.3 (Thermo Fisher Scientific, Bremen). Peak lists were searched using Mascot software version 2.3 (Matrix Science, UK) against a SwissProt database containing entries corresponding to Rattus norvegicus (version of July 2012), a list of common contaminants, and all the corresponding decoy entries. Trypsin was chosen as enzyme and a maximum of two miscleavages were allowed. Carbamidomethylation (C) was set as a fixed modification, whereas oxidation (M) and phosphorylation (STY) were used as variable modifications. Searches were performed using a peptide tolerance of 7 ppm, a product ion tolerance of 0.5 Da. Resulting data files were filtered for FDR less than 1 % at peptide level.

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Multi-modal comparison of molecular programs driving nurse cell death and clearance in Drosophila melanogaster oogenesis

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