Fig 4C raw data.sf3
MSMS analysis of immunoprecipitated OTULIN from HaCaT cells treated with TNF+CHX+zVAD for 6 hours.
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Mass Spectrometry: FLAG beads were digested with proteomics-grade Trypsin (Promega) at a concentration of 12 ng/ml. Digested peptides were extracted with 100 microliters of 100% Acetonitrile on a magnetic rack, the extract was transferred to a clean Eppendorf tube and dried in a Speed Vac for 1 hour. The dried peptides were then reconstituted in 25 miroliters of water supplemented with 0.1% formic acid (FA) and transferred to a 200 microliter sample vial. The peptide samples were subjected to LC reverse phase nanoflow chromatography using a Proxeon Easy nLC (Thermo Scientific). The peptides were trapped onto a 2 cm C18 trapping column (Acclaim PepMap 100, Thermo Scientific) and were separated at a flow rate of 350 nl/minute on a 15 cm C18 analytical nanocolumn (Acclaim PepMap RSLC, Thermo Scientific) with a water/Acetonitrile gradient covering 3%-38% Acetonitrile over 100 minutes. The eluting peptides were analyzed by an Orbitrap Q-Exactive HF (Thermo Scientific) operating with a duty cycle of 25 MSMS fragment spectra per precursor scan. The resolution was set at 120,000 (scan speed 2 spectra per second) for precursor scans, over mass range of 375-1400 m/z, and 30,000 (scan speed 25 spectra per second) for fragment spectra. The mass spectrometer was operated with a dynamic exclusion set at 6 seconds and maximum trap fill. Bioinformatics: The acquired spectra were converted into Mascot Generic Files (mgf) using Mascot Distiller (Matrix Sciences) and searched against the Human proteome (Uniprot). Searches were performed using the Mascot proteomics search engine (Matrix Sciences), setting mass tolerance of 5 ppm for precursor ions and 50 mDa for MSMS ions and selecting oxidized Methionine and phosphorylation on Serine, Threonine and Tyrosine as a variable modification. The searches were performed with a specified number of variable modifications. The Mascot data output was transferred to Scaffold (Proteome Software) for data validation. Redundant spectral counts were used as quantitative measure and spectra were accepted for peptides/proteins with a false discovery rate (FDR) of better than 5%.