Host defense by the innate immune system requires the establishment of antimicrobial states allowing cells to cope with microorganisms before the onset of the adaptive immune response. Interferons (IFN) are of vital importance in the establishment of cell-autonomous antimicrobial immunity. Speed is therefore an important attribute of the cellular response to IFN. With much of the antimicrobial response being installed de novo, this pertains foremost to gene expression, the rapid switch between resting-state and active-state transcription of host defense genes. Mechanisms to meet this demand on the relevant molecular machinery include remodeling of chromatin but also changes in transcription factor interaction prior and during the IFN response. Our results show how distinct transcription factor complexes, determine the responsiveness of Interferon stimulated genes to different IRF9-containing complexes. Raw 264.7 macrophages expressing a doxycycline-inducible, myc-tagged versions of each IRF9-BirA*, STAT2-BirA* and STAT1-BirA* fusion genes were used to study complex formation in vivo. Furthermore, we extended the BioID proximity labeling by coupling it to parallel reaction monitoring to determine the degree and quantity of association between IRF9 and STATs in resting and interferon-treated macrophages.

PRM assays were generated based on shotgun MS measurements acquired with the same MS instrument, selecting up to 10 high intensity proteotypic peptides for STAT1, STAT2, STAT3 and IRF9, with no missed cleavages, no methionine, and an even distribution over the chromatographic gradient. PRM assay generation was performed using Skyline. After a test run with a pooled sample showing high target protein expression, we reduced the targets to at least 5 peptides with a single charge state per protein by further optimising for best signal-to-noise and an even distribution over the gradient, resulting in a scheduled PRM assay with 4 min windows. Samples were spiked with 100 fmol Pierce Peptide Retention Time Calibration Mixture (PRTC, Thermo-Fisher) to monitor the chromatographic and nano-spray stability across the PRM measurements of all samples. Data analysis, manual validation of all peptides and their transitions (based on retention time, relative ion intensities, and mass accuracy), and relative quantification was performed in Skyline. The most intense non-interfering transition(s) of the top 5 peptides per protein were selected and their peak areas were summed up for peptide quantification (total peak area). Missing peptide intensities were imputed by random values derived from a normal distribution (down shift: median -2.15* standard dev., width: standard dev. *0.05). To correct for minor varying sample injection amounts and instrument stability over the measurements, MS1 signals of 6 stable background proteins were extracted selected based on an ANOVA analysis (q-value > 0.5, log2 intensity > 25 and standard dev. < 0.2 of MaxQuant LFQ intensities of the shot-gun measurements) and used to calculate normalization factors for the PRM dataset. After normalization, peptide intensity means were calculated for protein quantification. To ascertain significant interactions, mean log2 protein intensity ratios, standard deviation and t-test statistics were calculated for each of the target proteins.

BioID was performed according to a published protocol in three independent biological replicates per construct and condition. mycBioID was a gift from Kyle Roux (Addgene plasmid 35700). 5 x 106 stable Raw 264.7 cells were seeded on 15 cm dishes and treated with 0.2 µg/ml doxycycline for 24h. 50 µM biotin was added for 18 additional hours. Cells were either left untreated or stimulated for 2 h with IFN-beta or IFN-gamma or for 18 h with IFN-beta. Untreated controls were harvested at corresponding time points. Cells were washed and lysed at room temperature (lysis Buffer: 50 mMTris pH7.4; NaCL 500 mM; 0.2% SDS; EDTA 5mM + 1x protease inhibitors). Triton X-100 and 50 mMTris pH7.4 were added and the protein lysates were sonicated 2x for 30 seconds. Lysates were centrifuged for 5 minutes at full speed and supernatant were transferred to a new tube. Magnetic Pierce Streptavidin beads #88817 were washed 3x with lysis buffer. 105 µl beads were incubated with 1.3 mg of protein lysate over night at 4°C. 21 µl beads were kept for western blot analysis, the rest of the beads was used for the analysis with liquid chromatography mass spectrometry. Beads were washed at room temperature with wash buffer 1(2% SDS in H2O), wash buffer 2 (0.1% deoxycholic acid; 1% TritonX-100, 1mM EDTA, 500 mM NaCl, 50 mM HEPES; H2O) and wash buffer 3 (0.5% deoxycholic acid; 0,5% NP-40; 1 mM EDTA; 250 mM LiCl, 10 mM Tris pH7.4;H2O). Beads were washed 5 times with 50 mM Tris pH 7.4 and another two times with 50 mM ammonium bicarbonate (ABC) and then resuspended in 24 µL of 1 M urea in 50 mM ABC. 10 mM dithiothreitol (DTT) was added and the samples were incubated for 30 min at room temperature before adding 20 mM iodoacetamide and incubating for another 30 min at room temperature in the dark. Remaining iodoacetamide was quenched by adding 5 mM DTT and the proteins were digested with 300 ng (Trypsin Gold, Promega) at 37°C overnight. After stopping the digest by addition of 0.5% trifluoroacetic acid (TFA), and washing the beads with 30 µL 0.1% TFA, the supernatants were were loaded onto C18 stagetips to desalt the peptides prior to LC-MS. Peptides were separated on an Ultimate 3000 RSLC nano-flow chromatography system (Thermo-Fisher), using a pre-column for sample loading (Acclaim PepMap C18, 2 cm × 0.1 mm, 5 μm, Thermo-Fisher), and a C18 analytical column (Acclaim PepMap C18, 50 cm × 0.75 mm, 2 μm, Thermo-Fisher), applying a segmented linear gradient from 2% to 35% solvent B (80% acetonitrile, 0.1% formic acid; solvent A 0.1% formic acid) at a flow rate of 230 nL/min over 60 minutes. Eluting peptides were analyzed on a Q Exactive HF-X Orbitrap mass spectrometer (Thermo-Fisher), which was coupled to the column with a customized nano-spray EASY-Spray ion-source (Thermo-Fisher) using coated emitter tips (New Objective). For PRM data acquisition we used following MS parameters: survey scan with 60k resolution, AGC 1E6, 50 ms IT, over a range of 400 to 1300 m/z, PRM scan with 30k resolution, AGC 1E5, 300 ms IT, isolation window of 0.7 m/z with 0.2 m/z offset, and NCE of 27%.