MassIVE MSV000096618

Description

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. [doi:10.25345/C5R78616M] [dataset license: CC0 1.0 Universal (CC0 1.0)]

Keywords: Proximity labeling, Endoplasmic reticulum, Drosophila, Ovary ; DatasetType:Proteomics

Contact