Computational protocol: Compound A, a Selective Glucocorticoid Receptor Modulator, Enhances Heat Shock Protein Hsp70 Gene Promoter Activation

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[…] HEK293T cells were transiently transfected with 10µg Flag-tagged GR using the calcium phosphate transfection method. 24 hours after transfection and after induction with solvent or Compound A (10µM) for 3h, cells were lysed either in 1ml NP40 non-denaturing buffer (50 mM Tris.HCl pH 7.5; 125 mM NaCl; 5% glycerol; 0.2% NP40; 1.5 mM MgCl2; and cOmplete® protease inhibitor cocktail (1 tablet/50ml buffer)) or 1ml Buffer A (10mM Hepes pH 7.5; 10mM KCl; 1.5mM MgCl2; 0.1% NP-40; 0.5mM DTT; and for cOmplete® protease inhibitor cocktail cocktail (1 tablet/50ml buffer)). Input controls (50µl/sample) of either buffer were kept aside for control western blot analyses. Flag-tagged GR was immunoprecipitated with 40µl Flag beads overnight. Cell lysates in NP40 non-denaturing buffer were processed as follows: after 5 washes with 5mM TEAB (triethylammonium bicarbonate) buffer, elution was performed with 600 µl of 100 µg/ml Flag peptide in 5mM TEAB buffer. Cell lysates in buffer A were processed as follows: after 5 washes with NH4HCO3 (50mM pH 8), elution was done with 450 µl ammoniumhydroxide (NH4OH) (1%).Reagents and solvents were purchased from Sigma-Aldrich (St. Louis, MO, USA), except acetonitrile (Baker, Center Valley, PA, USA) and formic acid (Biosolve, Valkenswaard, The Netherlands), and used without further purification, unless specified. H2O for (HPLC) buffers was produced with a Millipore (Billerica, MA, USA) RIOs-DI water purification system, coupled with the MilliQ Reference A+ system equipped with a 22 µm filter and a C18 column to remove organic impurities.Immunoprecipated samples were dried completely in a rotational vacuum concentrator (RVC 2–33 IR, Martin Christ, Osterode am Harz, Germany). The residue was reconstituted in 500 µl of 50 mM TEAB buffer (pH 8) and the resulting solution was heated at 95°C for 10 min. After cooling on ice for at least 5 min, trypsin (2 µg, Sequencing Grade Modified Trypsin, Porcine, Promega, Leiden, The Netherlands) was added and the samples were overnight incubated at 37°C. Samples were again dried completely in a rotational vacuum concentrator and reconstituted in 20 µl of 2% acetonitrile in 0.1% TFA. Samples were centrifuged for 10 minutes at 15000 g, and the supernatant was transferred to a vial for LC-MS/MS analysis.The samples were analyzed on a LC-MS/MS system consisting of an Ultimate 3000 RSLC nano (Dionex, Amsterdam, The Netherlands) in-line connected to a LTQ Orbitrap Velos (Thermo Fisher Scientific, Bremen, Germany). The sample was loaded on a trapping column (made in-house, 100 µm internal diameter (I.D.)×20 mm, 5 µm beads C18 Reprosil-HD, Dr. Maisch). After back-flushing from the trapping column, the sample was loaded on a reverse-phase column (made in-house, 75 µm I.D×150 mm, 5 µm beads C18 Reprosil-HD, Dr. Maisch) with solvent A (0.1% trifluoroacetic acid, 2% acetonitrile), and were separated with a linear gradient from 2% solvent A’ (0.1% formic acid) to 55% solvent B’ (0.1% formic acid and 80% acetonitrile) at a flow rate of 300 nl/min followed by a wash reaching 100% solvent B.The mass spectrometer was operated in data-dependent mode, automatically switching between MS and MS/MS acquisition for the ten most abundant peaks in a given MS spectrum. Full scan MS spectra were acquired in the Orbitrap at a target value of 1E6 with a resolution of 60,000. The ten most intense ions were then isolated for fragmentation in the linear ion trap, with a dynamic exclusion of 30 s. Peptides were fragmented after filling the ion trap at a target value of 1E4 ion counts. The mass spectrometer’s nanospray source was expanded with an Active Background Ion Reduction Device (Abird, ESI Source Solutions). From the MS/MS data in each LC run, Mascot Generic Files were created using the Mascot Distiller software (version 2.4.3.3, Matrix Science, www.matrixscience.com/Distiller). While generating these peak lists, grouping of spectra was allowed in Distiller with a maximum intermediate retention time of 30 s and a maximum intermediate scan count of 5 was used where possible. Grouping was done with 0.005 m/z precursor tolerance. A peak list was only generated when the MS/MS spectrum contained more than 10 peaks. There was no de-isotoping and the relative signal to noise limit was set at 2. These peak lists were then searched with the Mascot search engine (MatrixScience) using the Mascot Daemon interface (version 2.4.0, Matrix Science). Spectra were searched against the human subsection of the Swiss-Prot database (version 2013_01 of UniProtKB/Swiss-Prot protein database containing 20,232 human sequence entries). Variable modifications were set to methionine oxidation, pyro-glutamate formation of amino terminal glutamine, and acetylation of the N-terminus and lysine side chains. The mass tolerance on precursor ions was set to 10 ppm (with Mascot’s C13 option set to 1), and on fragment ions to 0.5 Da. The peptide charge was set to 1+,2+,3+ and the instrument setting was put on ESI-TRAP. Enzyme was set to trypsin, allowing 1 missed cleavage, and cleavage was also allowed when arginine or lysine are followed by proline. Only peptides that were ranked one and scored above the threshold score, set at 99% confidence, were withheld. All data management was done by ms_lims . […]

Pipeline specifications

Software tools Mascot Distiller, Mascot Server, ms_lims
Databases UniProt UniProtKB
Application MS-based untargeted proteomics
Organisms Mus musculus