BUSCADOR RECOLECTA

Digital.CSIC. Repositorio Institucional del CSIC

oai:digital.csic.es:10261/310084

Data Files Description: NPC_XL_Identification_Inter_Crosslinked.csv: Inter-protein cross-links identified by pLink 2; NPC_XL_spectra.mgf: MS2 spectra data for the identified cross-links; NPC_XL_proteins.fasta : Protein sequences used for search., This repository contains chemical cross-linking mass spectrometry data of affinity-purified Yeast nuclear pore complexes., [Sample Processing] NPCs were immuno-purified from Mlp1 tagged S. cerevisiae strains (Kim et al., 2018). After native elution, 1.0 mM disuccinimidyl suberate (DSS) was added and the sample was incubated at 25ºC for 40 min with shaking (1,200 rpm). The reaction was quenched by adding a final concentration of 50 mM freshly prepared ammonium bicarbonate and incubating for 20 min with shaking (1,200 rpm) at 25ºC. The sample (50 µg) was then concentrated and denatured at 98ºC for 5 min in a solubilization buffer (10% solution of 1-dodecyl-3-methylimidazolium chloride (C12-mim-Cl) in 50 mM ammonium bicarbonate, pH 8.0, 100 mM DTT). After denaturation, the sample was centrifuged at 21,130 g for 10 min and the supernatant was transferred to a 100 kDa MWCO ultrafiltration unit (MRCF0R100, Microcon). The sample was quickly spun at 1,000 g for 2 min and washed twice with 50 mM ammonium bicarbonate. After alkylation (50 mM iodoacetamide), the cross-linked NPC in-filter was digested by trypsin and lysC O/N at 37ºC. After proteolysis, the sample was recovered by centrifugation and peptides were fractionated into 10-12 fractions by using a stage tip self-packed with basic C18 resins (Dr. Masch GmbH). Fractionated samples were pooled prior to LC/MS analysis. Desalted cross-link peptides were dissolved in the sample loading buffer (5% Methanol, 0.2% FA), separated with an automated nanoLC device (nLC1200, Thermo Fisher), and analyzed by an Orbitrap Q Exactive HFX (Pharma mode) mass spectrometer (Thermo Fisher) as previously described (Xiang et al., 2020; Xiang et al., 2021). Briefly, peptides were loaded onto an analytical column (C18, 1.6 μm particle size, 100 Å pore size, 75 μm × 25 cm; IonOpticks) and eluted using a 120-min liquid chromatography gradient. The flow rate was approximately 300 nl/min. The spray voltage was 1.7 kV. The QE HF-X instrument was operated in the data-dependent mode, where the top 10 most abundant ions (mass range 380 – 2,000, charge state 4 - 8) were fragmented by high-energy collisional dissociation (HCD). The target resolution was 120,000 for MS and 15,000 for tandem MS (MS/MS) analyses. The quadrupole isolation window was 1.8 Th; the maximum injection time for MS/MS was set at 200 ms., [Data processing] The raw data were searched with pLink2 (Chen et al., 2019b). An initial MS1 search window of 5 Da was allowed to cover all isotopic peaks of the cross-linked peptides. The data were automatically filtered using a mass accuracy of MS1 ≤ 10 ppm (parts per million) and MS2 ≤ 20 ppm of the theoretical monoisotopic (A0) and other isotopic masses (A+1, A+2, A+3, and A+4) as specified in the software. Other search parameters included cysteine carbamidomethyl as a fixed modification and methionine oxidation as a variable modification. A maximum of two trypsin missed-cleavage sites was allowed. The initial search results were obtained using a default 5% false discovery rate (FDR) expected by the target-decoy search strategy. Spectra were manually verified to improve data quality (Kim et al., 2018; Shi et al., 2014). Cross-linking data were analyzed and plotted with CX-Circos (http://cx-circos.net)., No