Quantitative Nanoproteomics for Protein Complexes (QNanoPX) Related to Estrogen Transcriptional Action
2009; Elsevier BV; Volume: 9; Issue: 2 Linguagem: Inglês
10.1074/mcp.m900183-mcp200
ISSN1535-9484
AutoresPai-Chiao Cheng, Hsiang-Kai Chang, Shu‐Hui Chen,
Tópico(s)Advanced Biosensing Techniques and Applications
ResumoWe 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 ERα but 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. 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 ERα but 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. Estrogen signaling is complex, involving two different isoforms of the estrogen receptor, α (ERα) 1The abbreviations used are:ERestrogen receptorQNanoPXquantitative nanoproteomics for resolving protein complexesAuNPgold nanoparticleEREestrogen response elementSAMself-assembly monolayerE217β-estradiolIPimmunoprecipitationPEGpolyethylene glycoldsEREdouble-stranded EREcEREcomplementary ERENHSN-hydroxysuccinimidyl. 1The abbreviations used are:ERestrogen receptorQNanoPXquantitative nanoproteomics for resolving protein complexesAuNPgold nanoparticleEREestrogen response elementSAMself-assembly monolayerE217β-estradiolIPimmunoprecipitationPEGpolyethylene glycoldsEREdouble-stranded EREcEREcomplementary ERENHSN-hydroxysuccinimidyl. and β (ERβ), as well as several different pathways that affect the expression of a number of genes either directly or indirectly. When activated by 17β-estradiol (E2), the ERs are translocated from the cytosol to the nucleus where the nuclear ERs bind to ERE and recruit other proteins in a complex by promoting, as an activator, or blocking, as a repressor, the recruitment of RNA polymerase to the target genes. The ER·ERE complex controls the transcription of genetic information from DNA to RNA as well as the translation from RNA to proteins. This process, which is known as the genomic pathway, is significantly involved with many diseases, including various cancers. A non-genomic pathway that involves membrane receptors and protein kinases to send the transduction signals to the nucleus has also been described (1Santen R.J. Song R.X. Zhang Z. Kumar R. Jeng M.H. Masamura S. Yue W. Berstein L. Adaptive hypersensitivity to estrogen: mechanism for superiority of aromatase inhibitors over selective estrogen receptor modulators for breast cancer treatment and prevention.Endocr.-Relat. Cancer. 2003; 10: 111-130Crossref PubMed Scopus (38) Google Scholar, 2Lupu R. Menendez J.A. Minireview: targeting fatty acid synthase in breast and endometrial cancer: an alternative to selective estrogen receptor modulators?.Endocrinology. 2006; 147: 4056-4066Crossref PubMed Scopus (100) Google Scholar). Although there have been studies involving proteomics profiling to identify estrogen-responsive proteins (3Zhu Z. Boobis A.R. Edwards R.J. Identification of estrogen-responsive proteins in MCF-7 human breast cancer cells using label-free quantitative proteomics.Proteomics. 2008; 8: 1987-2005Crossref PubMed Scopus (23) Google Scholar, 4Malorni L. Cacace G. Cuccurullo M. Pocsfalvi G. Chambery A. Farina A. Di Maro A. Parente A. Malorni A. Proteomic analysis of MCF-7 breast cancer cell line exposed to mitogenic concentration of 17 beta-estradiol.Proteomics. 2006; 6: 5973-5982Crossref PubMed Scopus (34) Google Scholar), the analysis of protein complexes based on a proteomics approach could provide more insights into specific signaling pathways and cross-interactions, which are rarely explored by other approaches.In recent years, the analysis of affinity-purified protein complexes in immunoprecipitation (IP) experiments coupled with a proteomics approach using tandem LC-MS/MS for the identification of proteins has become particularly attractive (5Matsuoka S. Ballif B.A. Smogorzewska A. McDonald 3rd, E.R. Hurov K.E. Luo J. Bakalarski C.E. Zhao Z. Solimini N. Lerenthal Y. Shiloh Y. Gygi S.P. Elledge S.J. ATM and ATR substrate analysis reveals extensive protein networks responsive to DNA damage.Science. 2007; 316: 1160-1166Crossref PubMed Scopus (2324) Google Scholar, 6Ranish J.A. Hahn S. Lu Y. Yi E.C. Li X.J. Eng J. Aebersold R. Identification of TFB5, a new component of general transcription and DNA repair factor IIH.Nat. Genet. 2004; 36: 707-713Crossref PubMed Scopus (124) Google Scholar). In principle, all the components, even of large complexes, can be identified in a single LC-MS experiment. Furthermore, quantitative proteomics approaches that are based upon stable isotope labeling, when performed along with appropriate control experiments, can distinguish background contamination or nonspecific binding from true interactors or differentiating effects that are caused by different biological states (7Wepf A. Glatter T. Schmidt A. Aebersold R. Gstaiger M. Quantitative interaction proteomics using mass spectrometry.Nat. Methods. 2009; 6: 203-205Crossref PubMed Scopus (119) Google Scholar, 8Liao L. Park S.K. Xu T. Vanderklish P. Yates 3rd, J.R. Quantitative proteomic analysis of primary neurons reveals diverse changes in synaptic protein content in fmr1 knockout mice.Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 15281-15286Crossref PubMed Scopus (139) Google Scholar, 9Boersema P.J. Aye T.T. van Veen T.A. Heck A.J. Mohammed S. Triplex protein quantification based on stable isotope labeling by peptide dimethylation applied to cell and tissue lysates.Proteomics. 2008; 8: 4624-4632Crossref PubMed Scopus (185) Google Scholar). Improvements in affinity purification that can be coupled with quantitative proteomics have also been developed, and most of these methods focus on the use of single/dual affinity tags (10Schulze W.X. Mann M. A novel proteomic screen for peptide-protein interactions.J. Biol. Chem. 2004; 279: 10756-10764Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar, 11Guerrero C. Tagwerker C. Kaiser P. Huang L. An integrated mass spectrometry-based proteomic approach: quantitative analysis of tandem affinity-purified in vivo cross-linked protein complexes (QTAX) to decipher the 26 S proteasome-interacting network.Mol. Cell. Proteomics. 2006; 5: 366-378Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar) or chemical reactions (12Phizicky E.M. Fields S. Protein-protein interactions: methods for detection and analysis.Microbiol. Rev. 1995; 59: 94-123Crossref PubMed Google Scholar), such as the use of in vivo cross-linking agents. In contrast, most of the IP assays are still performed using gel-coupled antibodies (12Phizicky E.M. Fields S. Protein-protein interactions: methods for detection and analysis.Microbiol. Rev. 1995; 59: 94-123Crossref PubMed Google Scholar). These gel beads have high binding capacity because of their porous nature. There are, however, major disadvantages also associated with the porous nature of gel beads composed of agarose or Sepharose. One such disadvantage involves a limitation on the ability of large complexes (13Alber F. Dokudovskaya S. Veenhoff L.M. Zhang W. Kipper J. Devos D. Suprapto A. Karni-Schmidt O. Williams R. Chait B.T. Sali A. Rout M.P. The molecular architecture of the nuclear pore complex.Nature. 2007; 450: 695-701Crossref PubMed Scopus (812) Google Scholar) to diffuse into the pores, which further renders an increase in nonspecific binding as more species could stick on the surface of the beads nonspecifically. Moreover, gel beads can precipitate quickly, which leads to incomplete interactions, even under continuous rotation. The >1-µm size of gel beads necessitates that a minimum quantity of beads be used for each experiment that is typically in the range of 25–50 µl of beads per IP. Monodispersed, superparamagnetic beads (14Cristea I.M. Williams R. Chait B.T. Rout M.P. Fluorescent proteins as proteomic probes.Mol. Cell. Proteomics. 2005; 4: 1933-1941Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar) in micro or nano sizes are available as a support material that could minimize sample loss and accelerate the processing speed via magnet-assisted separation. Magnetic beads, however, are likely to aggregate, possibly as a result of magnetism or non-homogeneous surface modifications, which therefore leads to incomplete recovery.Alternatively, gold nanoparticles (AuNPs) can easily be modified with a large selection of functional motifs by the use of self-assembly monolayer (SAM) technology to increase the solubility of the AuNPs, which could greatly improve interfacial interactions. These AuNPs could then be utilized in a variety of applications (15Baron R. Willner B. Willner I. Biomolecule-nanoparticle hybrids as functional units for nanobiotechnology.Chem. Commun. 2007; : 323-332Crossref PubMed Google Scholar). We previously demonstrated that monodispersed AuNPs are useful for concentrating proteins from a relatively large volume of dilute biological fluids by aggregation. This ability opens up new avenues of research because the traditional TCA precipitation method is ineffective under those conditions (16Wang A. Wu C.J. Chen S.H. Gold nanoparticle-assisted protein enrichment and electroelution for biological samples containing low protein concentrations: a prelude of gel electrophoresis.J. Proteome Res. 2006; 5: 1488-1492Crossref PubMed Scopus (31) Google Scholar). Modified AuNPs have been successfully used by other groups for the detection of DNAs (17Thaxton C.S. Georganopoulou D.G. Mirkin C.A. Gold nanoparticle probes for the detection of nucleic acid targets.Clin. Chim. Acta. 2006; 363: 120-126Crossref PubMed Scopus (288) Google Scholar) and proteins (18Aubin-Tam M.E. Hamad-Schifferli K. Structure and function of nanoparticle-protein conjugates.Biomed. Mater. 2008; 3 (034001)Crossref PubMed Scopus (205) Google Scholar) as well as for the fabrication of biosensors. In addition, the surface-only binding of AuNPs imposes no limitation on the size of protein complexes and eliminates the requirement for pore penetration, both of which are useful for IP experiments. Thus, AuNPs have several advantages that can be utilized to develop an efficient affinity purification method. Unlike nanomagnetic beads, AuNPs do need to be separated by centrifugation under conditions that require careful optimization. We investigate the protein-DNA interactome associated with ERE motifs located in the promoter region of a target gene. EREs are known to be regulated by ERα and ERβ, which are transcription factors that bind to the ERE itself. We proposed to functionalize AuNPs with the consensus sequence of ERE using the SAM technique and combine the affinity purification method with stable isotope dimethyl labeling (19Hsu J.L. Huang S.Y. Chow N.H. Chen S.H. Stable-isotope dimethyl labeling for quantitative proteomics.Anal. Chem. 2003; 75: 6843-6852Crossref PubMed Scopus (590) Google Scholar, 20Hsu J.L. Huang S.Y. Shiea J.T. Huang W.Y. Chen S.H. Beyond quantitative proteomics: Signal enhancement of the a(1) ion as a mass tag for peptide sequencing using dimethyl labeling.J. Proteome Res. 2005; 4: 101-108Crossref PubMed Scopus (106) Google Scholar, 21Hsu J.L. Huang S.Y. Chen S.H. Dimethyl multiplexed labeling combined with microcolumn separation and MS analysis for time course study in proteomics.Electrophoresis. 2006; 27: 3652-3660Crossref PubMed Scopus (59) Google Scholar), statistics, and informatics to identify the pulled down proteins that are associated with the ERE complex. This approach has been designated as quantitative nanoproteomics for protein complexes (QNanoPX). QNanoPX is expected to improve the ways that protein complexes can be analyzed by MS and to help resolve complexes that are related to the transcriptional action of estrogen. Estrogen signaling is complex, involving two different isoforms of the estrogen receptor, α (ERα) 1The abbreviations used are:ERestrogen receptorQNanoPXquantitative nanoproteomics for resolving protein complexesAuNPgold nanoparticleEREestrogen response elementSAMself-assembly monolayerE217β-estradiolIPimmunoprecipitationPEGpolyethylene glycoldsEREdouble-stranded EREcEREcomplementary ERENHSN-hydroxysuccinimidyl. 1The abbreviations used are:ERestrogen receptorQNanoPXquantitative nanoproteomics for resolving protein complexesAuNPgold nanoparticleEREestrogen response elementSAMself-assembly monolayerE217β-estradiolIPimmunoprecipitationPEGpolyethylene glycoldsEREdouble-stranded EREcEREcomplementary ERENHSN-hydroxysuccinimidyl. and β (ERβ), as well as several different pathways that affect the expression of a number of genes either directly or indirectly. When activated by 17β-estradiol (E2), the ERs are translocated from the cytosol to the nucleus where the nuclear ERs bind to ERE and recruit other proteins in a complex by promoting, as an activator, or blocking, as a repressor, the recruitment of RNA polymerase to the target genes. The ER·ERE complex controls the transcription of genetic information from DNA to RNA as well as the translation from RNA to proteins. This process, which is known as the genomic pathway, is significantly involved with many diseases, including various cancers. A non-genomic pathway that involves membrane receptors and protein kinases to send the transduction signals to the nucleus has also been described (1Santen R.J. Song R.X. Zhang Z. Kumar R. Jeng M.H. Masamura S. Yue W. Berstein L. Adaptive hypersensitivity to estrogen: mechanism for superiority of aromatase inhibitors over selective estrogen receptor modulators for breast cancer treatment and prevention.Endocr.-Relat. Cancer. 2003; 10: 111-130Crossref PubMed Scopus (38) Google Scholar, 2Lupu R. Menendez J.A. Minireview: targeting fatty acid synthase in breast and endometrial cancer: an alternative to selective estrogen receptor modulators?.Endocrinology. 2006; 147: 4056-4066Crossref PubMed Scopus (100) Google Scholar). Although there have been studies involving proteomics profiling to identify estrogen-responsive proteins (3Zhu Z. Boobis A.R. Edwards R.J. Identification of estrogen-responsive proteins in MCF-7 human breast cancer cells using label-free quantitative proteomics.Proteomics. 2008; 8: 1987-2005Crossref PubMed Scopus (23) Google Scholar, 4Malorni L. Cacace G. Cuccurullo M. Pocsfalvi G. Chambery A. Farina A. Di Maro A. Parente A. Malorni A. Proteomic analysis of MCF-7 breast cancer cell line exposed to mitogenic concentration of 17 beta-estradiol.Proteomics. 2006; 6: 5973-5982Crossref PubMed Scopus (34) Google Scholar), the analysis of protein complexes based on a proteomics approach could provide more insights into specific signaling pathways and cross-interactions, which are rarely explored by other approaches. estrogen receptor quantitative nanoproteomics for resolving protein complexes gold nanoparticle estrogen response element self-assembly monolayer 17β-estradiol immunoprecipitation polyethylene glycol double-stranded ERE complementary ERE N-hydroxysuccinimidyl. estrogen receptor quantitative nanoproteomics for resolving protein complexes gold nanoparticle estrogen response element self-assembly monolayer 17β-estradiol immunoprecipitation polyethylene glycol double-stranded ERE complementary ERE N-hydroxysuccinimidyl. In recent years, the analysis of affinity-purified protein complexes in immunoprecipitation (IP) experiments coupled with a proteomics approach using tandem LC-MS/MS for the identification of proteins has become particularly attractive (5Matsuoka S. Ballif B.A. Smogorzewska A. McDonald 3rd, E.R. Hurov K.E. Luo J. Bakalarski C.E. Zhao Z. Solimini N. Lerenthal Y. Shiloh Y. Gygi S.P. Elledge S.J. ATM and ATR substrate analysis reveals extensive protein networks responsive to DNA damage.Science. 2007; 316: 1160-1166Crossref PubMed Scopus (2324) Google Scholar, 6Ranish J.A. Hahn S. Lu Y. Yi E.C. Li X.J. Eng J. Aebersold R. Identification of TFB5, a new component of general transcription and DNA repair factor IIH.Nat. Genet. 2004; 36: 707-713Crossref PubMed Scopus (124) Google Scholar). In principle, all the components, even of large complexes, can be identified in a single LC-MS experiment. Furthermore, quantitative proteomics approaches that are based upon stable isotope labeling, when performed along with appropriate control experiments, can distinguish background contamination or nonspecific binding from true interactors or differentiating effects that are caused by different biological states (7Wepf A. Glatter T. Schmidt A. Aebersold R. Gstaiger M. Quantitative interaction proteomics using mass spectrometry.Nat. Methods. 2009; 6: 203-205Crossref PubMed Scopus (119) Google Scholar, 8Liao L. Park S.K. Xu T. Vanderklish P. Yates 3rd, J.R. Quantitative proteomic analysis of primary neurons reveals diverse changes in synaptic protein content in fmr1 knockout mice.Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 15281-15286Crossref PubMed Scopus (139) Google Scholar, 9Boersema P.J. Aye T.T. van Veen T.A. Heck A.J. Mohammed S. Triplex protein quantification based on stable isotope labeling by peptide dimethylation applied to cell and tissue lysates.Proteomics. 2008; 8: 4624-4632Crossref PubMed Scopus (185) Google Scholar). Improvements in affinity purification that can be coupled with quantitative proteomics have also been developed, and most of these methods focus on the use of single/dual affinity tags (10Schulze W.X. Mann M. A novel proteomic screen for peptide-protein interactions.J. Biol. Chem. 2004; 279: 10756-10764Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar, 11Guerrero C. Tagwerker C. Kaiser P. Huang L. An integrated mass spectrometry-based proteomic approach: quantitative analysis of tandem affinity-purified in vivo cross-linked protein complexes (QTAX) to decipher the 26 S proteasome-interacting network.Mol. Cell. Proteomics. 2006; 5: 366-378Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar) or chemical reactions (12Phizicky E.M. Fields S. Protein-protein interactions: methods for detection and analysis.Microbiol. Rev. 1995; 59: 94-123Crossref PubMed Google Scholar), such as the use of in vivo cross-linking agents. In contrast, most of the IP assays are still performed using gel-coupled antibodies (12Phizicky E.M. Fields S. Protein-protein interactions: methods for detection and analysis.Microbiol. Rev. 1995; 59: 94-123Crossref PubMed Google Scholar). These gel beads have high binding capacity because of their porous nature. There are, however, major disadvantages also associated with the porous nature of gel beads composed of agarose or Sepharose. One such disadvantage involves a limitation on the ability of large complexes (13Alber F. Dokudovskaya S. Veenhoff L.M. Zhang W. Kipper J. Devos D. Suprapto A. Karni-Schmidt O. Williams R. Chait B.T. Sali A. Rout M.P. The molecular architecture of the nuclear pore complex.Nature. 2007; 450: 695-701Crossref PubMed Scopus (812) Google Scholar) to diffuse into the pores, which further renders an increase in nonspecific binding as more species could stick on the surface of the beads nonspecifically. Moreover, gel beads can precipitate quickly, which leads to incomplete interactions, even under continuous rotation. The >1-µm size of gel beads necessitates that a minimum quantity of beads be used for each experiment that is typically in the range of 25–50 µl of beads per IP. Monodispersed, superparamagnetic beads (14Cristea I.M. Williams R. Chait B.T. Rout M.P. Fluorescent proteins as proteomic probes.Mol. Cell. Proteomics. 2005; 4: 1933-1941Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar) in micro or nano sizes are available as a support material that could minimize sample loss and accelerate the processing speed via magnet-assisted separation. Magnetic beads, however, are likely to aggregate, possibly as a result of magnetism or non-homogeneous surface modifications, which therefore leads to incomplete recovery. Alternatively, gold nanoparticles (AuNPs) can easily be modified with a large selection of functional motifs by the use of self-assembly monolayer (SAM) technology to increase the solubility of the AuNPs, which could greatly improve interfacial interactions. These AuNPs could then be utilized in a variety of applications (15Baron R. Willner B. Willner I. Biomolecule-nanoparticle hybrids as functional units for nanobiotechnology.Chem. Commun. 2007; : 323-332Crossref PubMed Google Scholar). We previously demonstrated that monodispersed AuNPs are useful for concentrating proteins from a relatively large volume of dilute biological fluids by aggregation. This ability opens up new avenues of research because the traditional TCA precipitation method is ineffective under those conditions (16Wang A. Wu C.J. Chen S.H. Gold nanoparticle-assisted protein enrichment and electroelution for biological samples containing low protein concentrations: a prelude of gel electrophoresis.J. Proteome Res. 2006; 5: 1488-1492Crossref PubMed Scopus (31) Google Scholar). Modified AuNPs have been successfully used by other groups for the detection of DNAs (17Thaxton C.S. Georganopoulou D.G. Mirkin C.A. Gold nanoparticle probes for the detection of nucleic acid targets.Clin. Chim. Acta. 2006; 363: 120-126Crossref PubMed Scopus (288) Google Scholar) and proteins (18Aubin-Tam M.E. Hamad-Schifferli K. Structure and function of nanoparticle-protein conjugates.Biomed. Mater. 2008; 3 (034001)Crossref PubMed Scopus (205) Google Scholar) as well as for the fabrication of biosensors. In addition, the surface-only binding of AuNPs imposes no limitation on the size of protein complexes and eliminates the requirement for pore penetration, both of which are useful for IP experiments. Thus, AuNPs have several advantages that can be utilized to develop an efficient affinity purification method. Unlike nanomagnetic beads, AuNPs do need to be separated by centrifugation under conditions that require careful optimization. We investigate the protein-DNA interactome associated with ERE motifs located in the promoter region of a target gene. EREs are known to be regulated by ERα and ERβ, which are transcription factors that bind to the ERE itself. We proposed to functionalize AuNPs with the consensus sequence of ERE using the SAM technique and combine the affinity purification method with stable isotope dimethyl labeling (19Hsu J.L. Huang S.Y. Chow N.H. Chen S.H. Stable-isotope dimethyl labeling for quantitative proteomics.Anal. Chem. 2003; 75: 6843-6852Crossref PubMed Scopus (590) Google Scholar, 20Hsu J.L. Huang S.Y. Shiea J.T. Huang W.Y. Chen S.H. Beyond quantitative proteomics: Signal enhancement of the a(1) ion as a mass tag for peptide sequencing using dimethyl labeling.J. Proteome Res. 2005; 4: 101-108Crossref PubMed Scopus (106) Google Scholar, 21Hsu J.L. Huang S.Y. Chen S.H. Dimethyl multiplexed labeling combined with microcolumn separation and MS analysis for time course study in proteomics.Electrophoresis. 2006; 27: 3652-3660Crossref PubMed Scopus (59) Google Scholar), statistics, and informatics to identify the pulled down proteins that are associated with the ERE complex. This approach has been designated as quantitative nanoproteomics for protein complexes (QNanoPX). QNanoPX is expected to improve the ways that protein complexes can be analyzed by MS and to help resolve complexes that are related to the transcriptional action of estrogen. Pathway analyses and data mining were done using the system provided by the Bioinformatics Core for Genomic Medicine and Biotechnology Development at the National Cheng-Kung University, supported by National Science Council Grant NSC 97-3112-B-006-011. Supplementary Material Download .zip (5.43 MB) Help with zip files Download .zip (5.43 MB) Help with zip files
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