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[…] Fragment mass spectra were converted into peaklists using the in-house software PAVA. HCD and ETD data were searched separately using ProteinProspector version 5.10.0 against the UniProt database with a concatenated database. Only mouse and human genomes were used for the database searching. Precursor tolerance was set to 10 ppm, whereas fragment mass error tolerance was set to 0.6 Da for ETD and 20 ppm for HCD. N-terminal acetylation, methionine oxidation, loss of N-terminal methionine and glutamate conversion to pyroglutamate were allowed as variable modifications. For ETD data, HexNAc modifications to serine and threonine residues and phosphorylation to serine/threonine/tyrosine was allowed as variable mass modifications. For HCD, phosphorylation was searched the same way though HexNAc was considered as a neutral loss. Methylation (mono, di- and tri-) of K and R, monomethylation of D, E and H (artifact from MeOH fixing PAGE gels), acetylation of K and R, and ADP-ribosylation to C, E, K, N, S, and R were searched separately. SLIP scoring was used to distinguish possible positional isomers of HexNAc and/or phosphopeptides (). Relative abundances of each modified or unmodified peptide were calculated using the ICIS area calculated from XICs in Xcalibur (Thermo Scientific) at a 10 ppm mass tolerance. [...] Six to ten 10 cm2 plates worth of haSOX2-Tg, fSOX2-Tg or fS248A-Tg cells were harvested for nuclear extract preparations in biological duplicate. Nuclear extracts were as previously described with minor modifications (). Buffers A, C and D were supplemented with 2 µM Thiamet G, 2 µM PUGNAc (Tocris Bioscience, United Kingdom) and 1X HALT protease and phosphatase inhibitors and instead of dialysis of extracts, two volumes of buffer D were used to dilute salt concentration. Seven µL of M2 beads per 10 cm2 plate were used per co-IP and samples were nutated at 4oC for two hours. Beads were washed once with Buffer D plus inhibitors, then twice with 50 mM ABC with 150 mM NaCl. Each wash was only as long as it took to transfer the beads to a new, cold tube and place on the magnetic rack. Beads were then resuspended in 50 µL 100 mM ABC with 500 ng trypsin and shaken at 37oC for one hour. Supernatant was transferred to a new tube, the beads were washed once with 50 µL ABC and combined to digest overnight. Digestions were desalted with one or two Zip Tips, depending on size of experiment, and dried via vacuum centrifugation. IP-WB experiments were performed similarly except proteins were eluted with 2X SDS-PAGE loading buffer without reducing agent.Chromatography was performed on a Nanoacquity HPLC (Waters) at 400 nl/min with an EASY-spray 15 cm x 75 µm ID, PepMap C18, 3 µm column (Thermo Scientific). A 90-minute gradient from 98% solvent A (0.1% formic acid) to 22% solvent B (0.1% formic acid in acetonitrile) was used. Peptides were analyzed on a Q-Exactive Plus mass spectrometer (Thermo Scientific). After the survey scan of m/z 400–1,600 was measured in the Orbitrap at 70,000 resolution, the top ten multiply charged ions were selected for HCD and measured at 17,500 resolution. Normalized collision energy for HCD was set at 35, Dynamic exclusion of precursor selection was set for 25 s.The label-free quantiation (LFQ) feature of MaxQuant (1.5.1.0) was used to quantify protein signals for proteins identified in the co-IP experiments. For SOX2, only non-TAD peptides were used for protein level quantitation. Proteins were determined to be SOX2 interactors by taking the average ratio of the LFQ intensity of the protein of interest (POI) from the FLAG-tagged mESC lines over the HA-tagged mEScs. This average ratio was log2-transformed, and was normalized by the global median. If the POI was two standard deviations from the mean it was considered specific to SOX2. Most proteins discussed were manually verified using Skyline.To determine POIs that differentially interact with wild type or S248A SOX2, we took the ratio of the log2-transformed ratio of FLAG/HA for S248A over the wild type. After normalization by the global median, POIs were considered to be differential interactors if they had a z-score of greater than 1.5. All of these peptides were manually verified using Skyline.For targeted analyses, FLAG-tagged MBD3 mESCs, a generous gift from the Fazzio laboratory () were analyzed as described above. From these analyses the top two most intense, non-homologous peptides identified from NuRD subunits were used to monitor their co-purification with SOX2 isoforms in fSOX2-Tg and fS248A-Tg cells in three separate, biological replicates. XICs were extracted manually using Xcalibur software. Relative enrichment of proteins co-purified with either SOX2 form was determined as described above. Peptides used for this analysis are listed in Supplementary Table 1b.Samples for co-IP Western blot corroboration of LC-MS data were performed as described above in biological duplicate. Antibodies used are described above. […]

Pipeline specifications

Software tools Protein Prospector, MaxQuant
Application MS-based untargeted proteomics
Organisms Mus musculus