DOI: 10.25345/c5tt4g50s
title: MassIVE MSV000095682 - MudPIT analyses of the proteins immunoprecipitated with the human Brg1 subunit of the BRG1/BRM-associated factor (BAF) SWI/SNF complex in hypoxic and normoxic conditions
title: Cell Culture | The HCT116 cell line was obtained from Horizon Discovery. Cells were cultivated in McCoy's Medium (Gibco) containing 10% FBS (Gibco), supplemented with 1% penicillin and streptomycin (Gibco) at 37oC in 5% CO2. The StemCell Technologies Hypoxia Incubator Chamber was used to generate a hypoxic environment. After 24 hrs cultivation in conventional cell culture incubator (allowing cells to attach to plates), plates were transferred to the chamber and 1% O2, 5% CO2, and 94% N2 gas mixture was purged into the chamber and placed into the conventional cell culture incubator for 24 hrs incubation. Antibody Conjugation to Protein A Beads | Approximately 2.5 ug of BRG1 antibody (Abcam) and 20 ul of Protein A Dynabeads (Invitrogen) were crosslinked at 4oC, rotating end over end for 4 hrs. Antibody-bound beads were washed 4X with ice cold PBS and once with 1 ml 0.1 M sodium borate (pH 9.0). Antibody-bound beads were resuspended with sodium borate with the addition of 5.2 mg of dimethylpyrimilidate (DMP) and allowed to rotate end over end at room temperature for 30 min. Antibody-bound beads were washed 2x with 0.2 M ethanolamine (pH 8.0) and then allowed to rotate end over end at 4oC overnight. Immunoprecipitation from Nuclear Extracts | Cells were seeded into 15 cm2 dishes with 8 x 10^6 cells. After 24 hrs, cells were incubated in normoxic or hypoxic conditions. Cells were harvested in ice-cold PBS. Approximately 1 mg of protein whole cell extract was added to Buffer C (20 mM HEPES, pH 7.9,1.5 mM MgCl2, 300 mM KCl, 0.2 mM EDTA, 10% glycerol, 0.1% NP-40) with complete protease inhibitors, 0.1 mM DTT and 1 mM PMSF and incubated on ice for 15 min, then spun at 14,000 rpm for 20 min at 4oC. Supernatant was removed and added to antibody-bound beads and incubated at 4oC, rotating end over end, for 4 hrs. Proteins immunoprecipitated from nuclear extracts were eluted in 0.1 M glycine (pH 2.5) for 2 min on ice with constant flicking of tube. Tris-HCl pH 8.8 was added to neutralize the pH. Liquid Chromatography Coupled to Tandem Mass Spectrometry | Endogenous immunoprecipitated proteins were TCA-precipitated overnight. After acetone washes, the protein pellet was solubilized using Tris-HCl pH 8.5 and 8 M urea, followed by addition of TCEP (Tris(2-carboxyethyl)phosphine hydrochloride; Pierce) and CAM (chloroacetamide; Sigma) were added to a final concentration of 5 mM and 10 mM, respectively. Proteins were digested using Endoproteinase Lys-C at 1:100 w/w (Roche) at 37oC overnight. The samples were brought to a final concentration of 2 M urea and 2 mM CaCl2 and a second digestion was performed overnight at 37oC using trypsin (Promega) at 1:100 w/w. The reactions were stopped with formic acid (5% final). The samples were loaded on a split-triple-phase fused-silica micro-capillary column and placed in-line with a linear ion trap mass spectrometer (LTQ, Thermo Scientific), coupled with a Quaternary Agilent 1100 Series HPLC system. A fully automated 10-step chromatography run was carried out. Each full MS scan (400-1600 m/z) was followed by five data-dependent MS/MS scans. Mass Spectrometry Data Analysis | RAW files were converted to .ms2 peak list files using RAWDistiller v.1.0. The MS2 dataset was searched using ProLuCID (v. 1.3.3) against a database consisting of 36631 Homo sapiens non-redundant proteins (NCBI, 2016-06-10 release), 194 usual contaminants, and, to estimate false discovery rates (FDRs), 36821 randomized amino acid sequences derived from each NR protein entry. To account for alkylation by CAM, 57 Da were added statically to the cysteine residues, while oxidation of methionine (+16 Da) was searched dynamically. The in-house software, swallow and sandmartin, were used in combination with DTASelect and Contrast (v. 1.9) to filter out peptide spectrum matches to maintain protein and peptides FDRs below 1%. The in-house NSAF7 software was used to calculate dNSAF values to quantify proteins by spectral counting and assemble results tables.