Discovery of Tamoxifen and N‑Desmethyl Tamoxifen Protein Targets in MCF‑7 Cells Using Large-Scale Protein Folding and Stability Measurements

• Published in: Journal of proteome research Vol. 16; no. 11; pp. 4073 - 4085
• Main Authors: Ogburn, Ryenne N, Jin, Lorrain, Meng, He, Fitzgerald, Michael C
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 03.11.2017
• Subjects: binding properties; binding proteins; breast neoplasms; Breast Neoplasms - chemistry; cell lines; estrogen receptors; Female; Humans; MCF-7 Cells; Molecular Targeted Therapy; oxidation; protein folding; Protein Folding - drug effects; Protein Stability - drug effects; proteolysis; proteome; Proteome - drug effects; proteomics; Proteomics - methods; Receptors, Estrogen - metabolism; tamoxifen; Tamoxifen - analogs & derivatives; Tamoxifen - pharmacology; Tamoxifen - therapeutic use; Y-Box-Binding Protein 1 - metabolism; proteomics; mode of action; off-target; iTRAQ; SPROX; mass spectrometry; SILAC; Y-Box binding protein 1; pulse proteolysis; chemical denaturation
• ISSN: 1535-3893; 1535-3907; 1535-3907
• DOI: 10.1021/acs.jproteome.7b00442

The proteins in an MCF-7 cell line were probed for tamoxifen (TAM) and n-desmethyl tamoxifen (NDT) induced stability changes using the Stability of Proteins from Rates of Oxidation (SPROX) technique in combination with two different quantitative proteomics strategies, including one based on SILAC and one based on isobaric mass tags. Over 1000 proteins were assayed for TAM- and NDT-induced protein stability changes, and a total of 163 and 200 protein hits were identified in the TAM and NDT studies, respectively. A subset of 27 high-confidence protein hits were reproducibly identified with both proteomics strategies and/or with multiple peptide probes. One-third of the high-confidence hits have previously established experimental links to the estrogen receptor, and nearly all of the high-confidence hits have established links to breast cancer. One high-confidence protein hit that has known estrogen receptor binding properties, Y-box binding protein 1 (YBX1), was further validated as a direct binding target of TAM using both the SPROX and pulse proteolysis techniques. Proteins with TAM- and/or NDT-induced expression level changes were also identified in the SILAC-SPROX experiments. These proteins with expression level changes included only a small fraction of those with TAM- and/or NDT-induced stability changes.