Proteomic Profiling of the TRAF3 Interactome Network Reveals a New Role for the ER-to-Golgi Transport Compartments in Innate Immunity

van Zuylen, Wendy J.; Doyon, Priscilla; Clément, Jean-François; Khan, Kashif Aziz; D'Ambrosio, Lisa M.; Dô, Florence; St-Amant-Verret, Myriam; Wissanji, Tasheen; Emery, Gregory; Gingras, Anne-Claude; Meloche, Sylvain; Servant, Marc J.

Preparation of whole cell extracts, co-immunoprecipitation studies, Native-PAGE and immunoblot analysis were performed as described previously [79] . A RIPA buffer (50 mM Tris-HCl, pH 7.4, 100 mM NaCl, 5 mM EDTA, 50 mM sodium fluoride, 40 mM b-glycerophosphate, 1 mM sodium orthovanadate, 1% Triton X-100, 0.1% SDS, 0.5% sodium deoxycholate, and protease inhibitors mixture (Sigma)) was used for the extraction of the TRAF3 AKKFF mutant. Antibodies were used as recommended by the manufacturers. FLAG affinity purification and mass spectrometric analysis FLAG-affinity purification was performed as described previously [80] with the following modifications. Detergent concentration in the lysis buffer was 0.5% NP-40; the lysis buffer was added at 4 ml/g wet cell pellet, and cells were subjected to passive lysis (30 minutes) followed by one freeze-thaw cycle and centrifugation. Immunoprecipitation was performed on the cleared lysate by adding 25 ml packed FLAG M2 beads (Sigma) and incubating for two hours. Beads were washed three times in lysis buffer, and three times in 50 mM ammonium bicarbonate. Samples were eluted with ammonium hydroxide, lyophilized in a speed-vac, resuspended in 50 mM ammonium bicarbonate (pH 8-8.3) , and incubated at 37uC with trypsin overnight. The ammonium bicarbonate was evaporated, and the samples were resuspended in HPLC buffer A2 (2% acetonitrile, 0.1% formic acid), then directly loaded onto capillary columns packed in-house with Magic 5 mm, 100A, C18AQ. MS/MS data was acquired in datadependent mode (over a 2 hr acetonitrile 2-40% gradient) on a ThermoFinnigan LTQ equipped with a Proxeon NanoSource and an Agilent 1100 capillary pump. Acquired RAW files were converted to mgf format using ProteoWizard. The searched database was human RefSeq (version 45). *.mgf files were searched with the Mascot search engine (version 2.3) using the following variable parameters: semi trypsin digestion, one missed cleavage allowed, asparagine deamidation and methionine oxidation. The fragment mass tolerance was 0.6 Da (monoisotopic mass), and the mass window for the precursor was +/23 Da (only +2 and +3 charge ions were processed). Mascot results were parsed for further analysis into a LIMS system developed at the Samuel Lunenfeld Research Institute [81] . Scoring of specific interactors for FLAG-TRAF3 and FLAG-p115 was performed using the statistical tool SAINT (Significance Analysis of INTeracome). SAINT converts label free quantification, such as spectral counts, for each prey protein identified in a purification of a bait into the probability of true interaction between the two proteins [82, 83] . SAINT can calculate a probability of interaction even for proteins proteins frequently detected in AP-MS experiments, providing that a quantitative enrichment is detected in the purification of the sample [84] . For each bait, two biological replicates were used. Twelve negative control runs (consisting of cells expressing the FLAG tag alone) were processed in parallel and combined into 5 virtual controls for SAINT modeling. SAINT calculates scores differently depending on the availability of negative control purifications, and thus the implementation for spectral count data incorporating control purification data was used (details are described in [82] ). The probability score was first computed for each prey in independent biological replicates separately (iProb). Then the final probability score for a pair of bait and prey proteins wa