br Data br Experimental design materials and methods br

Data
Experimental design, materials and methods
Acknowledgments This work was supported by grant no. 14-50-00068 from the Russian Science Foundation.
Data The proteomic profiling of atypical lung carcinoid (AC) tissue is presented, together with the largest collection of proteins identified from a large-cell neuroendocrine carcinoma (LCNEC) so far. A total of 1260 and 2436 proteins were identified in the AC and LCNEC samples, respectively, with FDR <1%. Noteworthy, these data have been obtained using archival formalin-fixed paraffin-embedded (FFPE) samples, thus reinforcing the opportunity and reliability of employing such samples for clinical proteomic studies. Data files can be found at PeptideAtlas repository at http://www.peptideatlas.org/PASS/PASS00375.
Experimental design, materials and methods Proteins were extracted from FFPE tissues as published [1], and quantified using the EZQ Protein Quantification Kit (Molecular Probes, Eugene, OR, USA). Detergent Removal Spin Columns (Pierce, Rockford, IL, USA) were used to remove SDS, and eluates were subjected to in solution reduction, alkylation and trypsin digestion as previously described [2]. ZipTip Pipette Tips (Millipore, Billerica, MA, USA) were employed to purify peptide mixtures. An LTQ Orbitrap Velos (Thermo Scientific, San Jose, CA, USA) interfaced with an UltiMate 3000 RSLCnano LC system (Dionex, Sunnyvale, CA, USA), set up in a data dependent MS/MS mode as described elsewhere [3], was used for MS analysis. Concentration, desalting, and separation of peptide mixtures was performed as in [2], but using a 270min gradient from 1 to 50% eluent B (0.2% formic GSK1324726A in 95% ACN) in eluent A (0.2% formic acid in 5% ACN). The gradient was as follows: 1–10% Solvent B (3min), 10–23% B (230min), 23–50% B (37min). Peptide identification was performed using Proteome Discoverer (version 1.4; Thermo Scientific) as described previously [2].
Acknowledgments This work was financed by Sardegna Ricerche through the program “Progetto Strategico Biotecnologie”, by AIRCIG 2013 14696 to GR, and by grant 997/2010.0414 from Fondazione Banco di Sardegna to GF.
Value of the data
Data Here, we present FTS data featuring multidrug-binding nature of the transcriptional regulator StAcrR. The top sixteen binders of StAcrR, as shown in Table S1, were identified by the FTS analysis from a library of 320 unique ligands. Human enolase 1, a negative control protein (Fig. S1), did not bind the top six binders of StAcrR, while implying their specific interactions with StAcrR. The remaining compounds were not tested against human enolase 1. Theoretical data for Table S2 were obtained from National Center for Biotechnology Information PubChem Compound Database (https://pubchem.ncbi.nlm.nih.gov/compound/) and include chemical properties such as topological polar surface (Å3), number of hydrogen bond donors/acceptors, XLogP3, and molecular weight (g/mol) of the top six StAcrR FTS hits, dequalinium chloride, proflavine, ethidium bromide and rhodamine 6G. The latter three molecules were identified as the StAcrR binders employing a fluorescence polarization experimental approach (see our original publication [1]).
Experimental design, materials and methods The FTS assay was run in 384-well PCR plates using an Echo550 acoustic transfer robot (Labcyte, Sunnyvale, CA) for dispensing a dimethyl sulfoxide stock of ligands to assay plates that contain 10μl of a mixture of StAcrR (1μg) and 2.5×SYPRO Orange fluorescence dye (Invitrogen, Carlsbad, CA) in 100mM HEPES buffer pH 7.5 and 150mM NaCl. Thermal scanning (from 10 to 80°C at 1.5°C min−1 ramp rate) on a real-time PCR machine CFX384 (Bio-Rad Laboratories, Hercules, CA) was coupled with fluorescence detection every 10s. A test molecule, dequalinium, bound at 40μM that prompted us to screen a 320-molecule subset of the Spectrum library (Micro Source Discovery, Gaylordsville, CT, hereafter referred as SPC2-ECH008), dequalinium belongs to. The best binders were selected based on ΔTm, reduction of the background reading, and shape of the melting curve. The top six hits were further subjected to a dose-dependent response analysis using their 2.5, 5, 10, 25, 50, 75 and 100μM concentrations. Human enolase 1 (1.2μg in 10μl assay mixture) was used as a negative control protein and tested against the top six binders at 10, 25 and 50 100μM concentrations. FTS data were analyzed with the in-house ExcelFTS software.