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[…] SILAC heavy and light collected serum-free cell media were mixed proportionally to a 1:1 cell number ratio and lysed with 8 M Urea 10 mM Tris-HCl buffer. Proteins were then quantified by Bradford. About 5 µg of mixed proteins for each sample were reduced by TCEP, alkylated by chloroacetamide, and digested by Lys-C and trypsin (), before peptides were desalted on StageTip C18 ().Samples were analyzed in duplo on a LC–ESI–MS-MS quadrupole Orbitrap QExactive-HF mass spectrometer (Thermo Fisher Scientific). Peptides separation was achieved on a linear gradient from 93% solvent A (2% ACN, 0.1% formic acid) to 60% solvent B (80% acetonitrile, 0.1% formic acid) over 110 min,and from 60% to 100% solvent B in 8 min at a constant flow rate of 0.25 µl/min on UHPLC Easy-nLC 1000 (Thermo Fischer Scientific) connected to a 23 cm fused-silica emitter of 75 µm inner diameter (New Objective, Inc. Woburn, MA, USA), packed in-house with ReproSil-Pur C18-AQ 1.9 µm beads (Dr Maisch Gmbh, Ammerbuch, Germany) using a high-pressure bomb loader (Proxeon, Odense, Denmark). MS data were acquired using a data-dependent top 20 method for HCD fragmentation. Survey full scan MS spectra (300–1650 Th) were acquired in the Orbitrap with 60,000 resolution, AGC target 3e6, IT 20 ms. For HCD spectra, resolution was set to 15,000 at m/z 200, AGC target 1e5, IT 80 ms; NCE 28% and isolation width 2.0 m/z.For identification and quantitation, raw data were processed with MaxQuant version 1.5.2.8 searching against the database uniprot_cp_human_2015_03 + sequences of µs and the Mif responsive hybrid nuclear receptor Switch setting labeling Arg10 and Lys8, trypsin specificity and up to two missed cleavages. Cysteine carbamidomethyl was used as fixed modification, methionine oxidation and protein N-terminal acetylation as variable modifications. Mass deviation for MS-MS peaks was set at 20 ppm. The peptides and protein false discovery rates (FDR) were set to 0.01; the minimal length required for a peptide was six amino acids; a minimum of two peptides and at least one unique peptide were required for high-confidence protein identification.The lists of identified proteins were filtered to eliminate known contaminants and reverse hits. Normalized H/L ratios were analyzed via Perseus (version 1.5.0.6). Statistical analysis was performed using the Significance B outlier test where statistical significance based on magnitude fold-change was established at p<0.05. To look for proteins that changed over time, we considered Intensity L and Intensity H normalized by the correspondent Summed Intensity and the statistical ANOVA test analysis was performed using Perseus (ver. 1.5.0.6) with p<0.01. All proteomic data as raw files, total proteins, and peptides identified with relative intensities and search parameters have been loaded into Peptide Atlas repository (ftp://PASS01009:[email protected]/). To obtain approximations for absolute protein quantities, we followed a MaxLFQ label-free quantification strategy, as described previously (). We then assessed the relative abundance of total protein per subcellular compartment, that is cytosol, nucleus, mitochondrion, ER, and other organelles, as well as µs, as well as the relative abundance of protein species, including or excluding µs, within the ER as detailed in the legend of . […]

Pipeline specifications

Software tools MaxQuant, Perseus
Application MS-based untargeted proteomics