Quantitative Nano-Proteomics for Protein Complexes (QNanoPX) Related to Estrogen Transcriptional Action

• Main Authors: Pai-ChiaoCheng, 鄭百喬
• Other Authors: Shu-Hui Chen
• Format: Others
• Language: en_US
• Published: 2010
• Online Access: http://ndltd.ncl.edu.tw/handle/80870694996927644302

博士 === 國立成功大學 === 化學系碩博士班 === 98 === We developed an integrated proteomics approach using a chemically functionalized gold nanoparticle (AuNP) as a novel probe for affinity purification in order to analyze a large protein complex in vivo. We then applied this approach to globally map the transcriptional activation complex of the estrogen response element (ERE). This approach was designated as Quantitative Nano-Proteomics for Protein complexes (QNanoPX). In this approach, the positive AuNP-ERE probes were functionalized with polyethylene glycol (PEG) and the consensus sequence of ERE and negative AuNP-PEG probes were functionalized with PEG without the ERE via thiolated self-assembly monolayer (SAM) technique. The AuNP-ERE probe had substantially low nonspecific binding and high solubility, which resulted in a 20-fold enrichment of the factor compared to gel beads. In addition, the surface-only binding allows the probe to capture a large protein complex without any restrictions due to pore size. The affinity purification method was combined with MS-based quantitative proteomics and statistical methods to reveal the components of the ERE complex in MCF-7 cells and to identify those components within the complex that were altered by the presence of 17?-estradiol (E2). Results indicated that a majority of proteins pulled down by the positive probe exhibited significant binding, and approximately one-half of the proteins, including estrogen receptor ? (ER?), were slightly but significantly affected by a 24-h treatment with E2. Based on a combination of bioinformatic and pathway analysis, most of the affected proteins, however, appeared to be related to the transcriptional regulation of not only ER?, but also c-Myc. Further confirmation indicated that E2 enhanced the ERE binding of c-Myc by 14-fold, which indicated that c-Myc may play a major role, along with ER?, in E2-mediated transcription. Taken together, our results demonstrated a successful QNanoPX approach towards pathway discovery and further revealed the importance of cross-interactions among transcription factors.