Quantitative nanoproteomics for protein complexes (QNanoPX) related to estrogen transcriptional action

Pai Chiao Cheng, Hsiang Kai Chang, Shu-Hui Chen

Research output: Contribution to journalArticle

26 Citations (Scopus)

Abstract

We developed an integrated proteomics approach using a chemically functionalized gold nanoparticle (AuNP) as a novel probe for affinity purification 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 nanoproteomics 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 a thiolated self-assembly monolayer technique. The AuNP-ERE probe had substantially low nonspecific binding and high solubility, which resulted in a 20-fold enrichment of the factor compared with 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 17β-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 α (ERα), were slightly but significantly affected by a 24-h treatment with E2. Based on a combination of bioinformatics and pathway analysis, most of the affected proteins, however, appeared to be related to the transcriptional regulation of not only ERαbut also c-Myc. Further confirmation indicated that E2 enhanced the ERE binding of c-Myc by 14-fold, indicating that c-Myc may play a major role, along with ERα, in E2-mediated transcription. Taken together, our results demonstrated a successful QNanoPX approach toward new pathway discovery and further revealed the importance of cross-interactions among transcription factors.

Original languageEnglish
Pages (from-to)209-224
Number of pages16
JournalMolecular and Cellular Proteomics
Volume9
Issue number2
DOIs
Publication statusPublished - 2010 Feb 1

Fingerprint

Response Elements
Estrogens
Proteins
Estrogen Receptors
Proteomics
Purification
MCF-7 Cells
Consensus Sequence
Transcription
Bioinformatics
Computational Biology
Gold
Nanoparticles
Solubility
Self assembly
Transcriptional Activation
Pore size
Estradiol
Monolayers
Statistical methods

All Science Journal Classification (ASJC) codes

  • Analytical Chemistry
  • Biochemistry
  • Molecular Biology

Cite this

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abstract = "We developed an integrated proteomics approach using a chemically functionalized gold nanoparticle (AuNP) as a novel probe for affinity purification 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 nanoproteomics 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 a thiolated self-assembly monolayer technique. The AuNP-ERE probe had substantially low nonspecific binding and high solubility, which resulted in a 20-fold enrichment of the factor compared with 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 17β-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 α (ERα), were slightly but significantly affected by a 24-h treatment with E2. Based on a combination of bioinformatics and pathway analysis, most of the affected proteins, however, appeared to be related to the transcriptional regulation of not only ERαbut also c-Myc. Further confirmation indicated that E2 enhanced the ERE binding of c-Myc by 14-fold, indicating that c-Myc may play a major role, along with ERα, in E2-mediated transcription. Taken together, our results demonstrated a successful QNanoPX approach toward new pathway discovery and further revealed the importance of cross-interactions among transcription factors.",
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Quantitative nanoproteomics for protein complexes (QNanoPX) related to estrogen transcriptional action. / Cheng, Pai Chiao; Chang, Hsiang Kai; Chen, Shu-Hui.

In: Molecular and Cellular Proteomics, Vol. 9, No. 2, 01.02.2010, p. 209-224.

Research output: Contribution to journalArticle

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