Mass spectrometry data for manuscript "RBM25 selectively stabilizes XBP1s mRNA independently of splicing factor activity"

Intro Aim of the experiment was to identify proteins interacting with 3'UTRs of XBP1 mRNA. Potential binding partners were affinity copurified from cytosolic fraction of cellular extracts using biotinilated mRNA baits. Sample legend batch 1 241205_ZBF_AWI_3.raw; spliced XBP1 3'UTR mRNA bait; tunicamycin treated cells 241205_ZBF_AWI_4.raw; unspliced XBP1 3'UTR mRNA bait; tunicamycin treated cells 241205_ZBF_AWI_N3.raw; spliced XBP1 3'UTR mRNA bait; untreated cells 241205_ZBF_AWI_N4.raw; unspliced XBP1 3'UTR mRNA bait; untreated cells batch 2 250124_ZBF_AWI_3T_MSe.raw; spliced XBP1 3'UTR mRNA bait; tunicamycin treated cells 250124_ZBF_AWI_4T_MSe.raw; unspliced XBP1 3'UTR mRNA bait; tunicamycin treated cells 250124_ZBF_AWI_3N_MSe.raw; spliced XBP1 3'UTR mRNA bait; untreated cells 250124_ZBF_AWI_4N_MSe.raw; unspliced XBP1 3'UTR mRNA bait; untreated cells Filename legend 241206_ZBF_AWI_qip_proteins.csv; batch 1 QI output protein list 250127_ZBF_AWI_qip_proteins.csv; batch 2 QI output protein list raw.zip archives are MassLynx v4.2 raw files from each acquistion 7z archives are open-vendor format spectra (.mgf) and identifications (.mzid) from each batch RNA-bait pull-down HeLa S3 cells were cultured in 100 mm dishes (10 dishes per condition; no stress (NS) or Tm-treated). Cells were washed twice with ice cold PBS (Biowest, L0616), scraped into 1 ml PBS per dish, and collected by centrifugation (5 min, 300 × g, 4 °C). After removing PBS, the packed cell volume (PCV) was determined. Cell pellets were resuspended in five volumes of lysis buffer (10 mM HEPES, 10 mM KCl, 1 mM MgCl₂, 1 mM DTT, and 1x Protease Inhibitor cOmplete Mini (11836170001; Roche), pH 7.4) and incubated on ice for 20 min. Cells were lysed by passing the suspension 20 times through a syringe needle (G27). Lysates were clarified by ultracentrifugation (1 h, 100,000 × g, 4 °C), and protein concentration of the resulting cytoplasmic fraction was determined by A₂₈₀ absorbance.For RNA pulldown assays, 1 µg of biotinylated RNA bait was incubated with 500 µg of cytoplasmic extract for 1 h at room temperature in the presence of 20 U RiboLock. Streptavidin magnetic beads (50 µl; Pierce, 88816) were added and incubated for an additional 1 h at room temperature with gentle agitation. Beads were collected using a magnetic rack and washed twice with wash buffer (0.6 M NaCl, 20 mM Tris, 10 mM EDTA, 1 mM DTT, 1× PIC, pH 7.4). Mass spectrometry After the final wash, resin-bound proteins were denatured for 10 mins. in 65°C in presence of 0.5% sodium deoxycholate (DOC:Na, Merck). Next, 100 ng of Trypsin (EMS0006, Merck) was added to the samples for an overnight digestion in 37°C. On the following day, the peptide solution was separated from the resin, DOC:Na was removed via acidification and centrifugation, and the supernatants underwent desalting with the STAGE tip protocol. The obtained peptide pellet was resuspended in a 0.1% formic acid (FA) + 3% acetonitrile (ACN) solution.LC-MS was carried out on an M-Class Acquity nanoUPLC system coupled to a Synapt XS HDMS equipped with a nanoESI ion source interface. Mobile phase A consisted of H2O + 0.1% FA, while mobile phase B of ACN + 0.1% FA. Samples were separated on an analytical column (nanoEase HSS T3 C18 100Å, 1.8μm, 75μm x 150mm) kept at 40°C with a 40min linear gradient of 5–35%B at a 300nL/min flow rate. A 3min trapping step was performed prior to gradient separation. Injected volume was 3µL. MS data were collected in DIA mode, scanning at 2Hz through a 50–2000m/z range in ESI+ and analyser mode set to Resolution (25000 FWHM at 785.84m/z). For MS2 of all ions, a collision energy ramp of 25–45V was set on instrument’s trap cell. Source conditions were fine-tuned and kept constant during acquisitions. A Glu-Fib (Waters) solution was acquired in the mass reference function, and the correction was applied post-acquisition.Raw processing was performed using Progenesis QI for Proteomics v4.2.7 (Non-linear Dynamics). QI’’s autovalues were unchanged for the deconvolution, and the set low and high energy ion thresholds were 200 and 20, respectively. Deconvoluted data were searched via Ion Accounting against a redundant human protein sequence databank (UP000005640, 83526 sequences) appended with the CCP version of cRAP contaminant sequences. The search parameters were as follows; peptide/fragment mass tolerance: 10/20 ppm; min. fragments/peptide: 3; min. fragments/protein: 7; min. peptides/protein: 1; max. protein mass: 1MDa; enzyme: trypsin; max. missed cleavages: 4; variable modification: oxidation of methionine; FDR: 4% (protein-level). Protein grouping was enabled, and relative quantitation was set according to non-conflicted peptides. Lists containing uniquely identified and quantified proteins from two independent experiments (SF1, SF2) were exported for further analysis.