Comparative proteomic data of M13SV1 human breast epithelial cells and their tumorigenic variants under treatment with estrogenic compounds

Data from a comparative proteomic analysis of three human breast epithelial cell lines are presented. M13SV1 cells and their tumorigenic derivatives M13SV1-R2-2 and M13SV1-R2-N1 were used. Proteomic data were obtained using 2-dimensional gel electrophoresis and subsequent identification of proteins by MALDI-TOF mass spectrometry. In a second experiment, the three cell lines were treated with different concentrations of the estrogenic compounds β-estradiol or genistein and alterations in protein expression were monitored by 2-dimensional gel electrophoresis and MALDI-TOF mass spectrometry. Presented data provide a comprehensive overview of proteomic differences between the three cell lines and their response to estrogenic stimulation.


Figures, protein expression tables How data was acquired
Cell culture, 2-dimensional gel electrophoresis, MALDI-TOF mass spectrometric identification of proteins Data format Normalized data.

Experimental factors
Cell line identity, treatment with genistein or β-estradiol, 2D/MS proteome analysis Experimental features M13SV1 human breast epithelial cells and their tumorigenic variants M13SV1-R2-2 and M13SV1-R2-N1 were treated with different concentrations of genistein or β-estradiol. Protein extracts were separated by 2-dimensional gel electrophoresis. Following identification of significantly altered protein spots, spots were picked and identified using MALDI-TOF mass spectrometry.

Data source location
German Federal Institute for Risk Assessment, Berlin, Germany Data accessibility Data is with this article.

Value of the data
Differences between M13SV1 human breast epithelial cells and their tumorigenic variants M13SV1-R2-2 and M13SV1-R2-N1 were identified by comprehensive proteomic analysis Quantitative proteomic responses of the three cell lines to the estrogenic compounds genistein and β-estradiol were identified Data can help to elucidate biological mechanisms of tumorigenicity and the molecular response of breast cancer cells to estrogenic substances.

Data
Proteomic profiles of the three cell lines M13SV1 human breast epithelial cells [1] and their tumorigenic variants M13SV1-R2-2 and M13SV1-R2-N1 [2] were obtained by 2-dimensional gel electrophoresis followed by identification of significantly differentially expressed protein spots by MALDI/TOF mass spectrometry. In addition, the proteomic responses of the three cell lines to treatment with different concentrations of the estrogenic compounds genistein and β-estradiol were analyzed.
Data are presented in form of an Excel spreadsheet (supplementary file) containing information about the identity of the proteins, fold difference in abundance between different cell lines or different treatments, and corresponding p values. Table 1 of the supplementary file contains the data of a comparison of cell lines, i.e. proteins differentially expressed in the tumorigenic cell lines M13SV1-R2-2 (termed "B" throughout the supplementary file) and M13SV1-R2-N1 (termed "C"), as compared to the non-tumorigenic line M13SV1 (termed "A"). Tables 2-4 of the supplementary file contain the data of proteins de-regulated in cell lines A-C following treatment with genistein (0.1 mM, 1 mM, 10 mM; termed "G1-G3") or β-estradiol (10 À 5 mM, 10 À 3 mM, 1 mM; termed "E1-E3"). A legend with explanations is contained in the last tab, termed "explanations". Please see also Fig. 1 for an overview of cell lines and treatment regimen.

Two-dimensional gel electrophoresis
Two-dimensional gel electrophoresis has been described in detail in a previous paper [3]. For details on the experimental setup, please refer to the latter publication. In brief, isoelectric focusing using IPG strips (pH 3-10) was followed by SDS-PAGE in a 12.5% acrylamide gel. Spots were stained with Tris Ruthenium II. Four gels (technical replicates) were run per sample, in biological triplicates for each experimental condition. Protein spots were identified and quantified using a VersaDoc imaging system and ProteinMine 1.6.1 software.

Statistical evaluation of gels
Data analysis and statistical evaluation of 2D gels using the software R has been described in detail in a previous paper [3]. For details on the experimental setup, please refer to the latter publication. A normalization of 2-DE gel images was performed using the total spot volume, related to an anchor template gel image as reference. Spot intensity values, given as sum of pixel intensities of each spot area, were used as a measure for protein spot quantification. Only spots quantified in at least two out of three biological and two out of four technical replicates were considered for statistical analysis. Comparison of treatment groups was done using a two-sided Wilcoxon rank sum test and statistical significance was assumed at po 0.05). A cutoff of │log 2 ratio│ Z0.3 for up-or downregulation of individual protein spots was applied.

Spot picking and in-gel digestion
Robot-assisted spot picking and tryptic in-gel digestion methodology has been described in detail in a previous paper [3]. For details on the experimental setup, please refer to the latter publication. Approximately 3000 spots were identified per gel. For an overview of the number of deregulated protein spots in the cell line comparison and the estrogenic cell treatment experiment, please refer to Fig. 2 and Fig. 3.

Mass spectrometry
Mass spectrometric analyses were used for protein identification, whereas protein quantification was based on spot intensity evaluation of 2D gel electrophoresis (see also section 2.2). Protein identification by mass spectrometry has been described in detail in a previous paper [3]. For details on the experimental setup, please refer to the latter publication. In brief, digested proteins were manually spotted on an AnchorChip Target (800/384, Bruker) and matrix solution was added. An Ultraflex II mass spectrometer (Bruker) with smartbeam laser was used for matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analysis with a mass range of 500-5000 Da. For more details on mass spectrometer settings, please refer to [3]. Flex Control and Flex Analysis software (version 3.0, Bruker) were used to process MS and MS/MS spectra. Protein identification was performed using Biotools software (version 3.2, Bruker) with Mascot search using the SwissProt database (accessed between January 09th and June 17th, releases 2015_01 À 2016_06; taxonomy: homo sapiens). For each protein to identify, two protein spots were digested and measured independently using peptide mass fingerprinting and MS/MS, giving 4 results. Only proteins identified with a minimum of 2 hits were considered valid. The column header "hits" in the Supplementary file shows the number of identifications for the two spot measurements. Sequence coverage of each identified protein is also indicated in the Supplementary file.