Investigation of Tumor Differentiation Factor (TDF)‐induced Cell Differentiation using Mass Spectrometry

Abstract

Tumor differentiation factor is a protein, produced by the pituitary gland and is secreted into the bloodstream. TDF is known to induce differentiation in human breast and prostate cancer cells and not in other cells. However, the site of action or receptor and the mechanism of action of TDF to induce differentiation are unknown. In recent studies done by our group, potential TDF receptor candidates in human breast and prostate cancer cells were identified. Substantial differences were also observed in the SDS‐PAGE protein patterns of untreated and cancer cells treated with TDF analogs. Hence, the current study is focused on the proteomic analysis of TDF‐untreated and TDF treated MCF7 and DU145 human breast and prostate cancer cells, on functional investigation of TDF as well as on the localization of TDF. These experiments will help elucidate the mechanism through which TDF induces cell differentiation. To carry out these investigations, MCF‐7 human breast and DU‐145 human prostate cancer cells were grown under standard cell culture conditions. The cells were treated with various concentrations of TDF analogs(0 to 10 µg), lysed and the proteins were separated by SDS‐PAGE and stained with Coomassie for qualitative inspection and direct comparison of the samples. The gel bands were cut into pieces which were then digested and the different peptide mixtures were analyzed by nanoliquid chromatography‐tandem mass spectrometry (nanoLC‐MS/MS) using a nanoAcquity UPLC coupled with a QTOF Micro mass spectrometer, followed by data analysis using MassLynx (version 4.1), ProteinLynx Global Served (PLGS 2.4), Mascot and Scaffold 3.0. Further validation and functional studies will also be performed.