乳癌的發生率在全世界都非常高，尤其是女性獲得乳癌的機率也相對較男性來的高，衛生署去年度的統計資料，女性乳癌的排名位居第四，而雌激素不只是對乳房細胞有影響，對骨頭及泌尿系統也會有作用，當然最明顯的影響就是對上乳房細胞會有促進分化生長的癌化現象，所以如何利用金奈米粒子載體，針對乳癌細胞中的雌激素受體進行純化濃縮，將在後續的實驗對於雌激素探針和受體進行探討。
利用金奈米粒子作為固態載體，並配合類固醇的雌激素小分子 (E2-biotin)，利用 biotin-avidin 系統，對乳癌細胞 (MCF-7) 細胞萃取液中的雌激素受體蛋白的其中一個構型 (ERα) 進行親和性作用而被金奈米粒子純化。在實驗當中以含有 E2-biotin 作為實驗組，控制組則是不含雌激素衍生物，利用非直接修飾在奈米金粒子上的雌激素衍生物，降低系統中的非專一性吸附蛋白，並將得到的目標蛋白經過酵素消化後，利用高壓液相層析質譜儀，辨認與雌激素受體相關的蛋白質，再經由蛋白質資料庫比對胜肽序列，提供在化學蛋白質體學上的一個新視野。

The most common cancer affecting women is breast cancer. Estrogens regulate multiple cellular function via estrogen receptor alpha (ERα). In order to have more information about various biological events by the estrogen binding position and surface character with ERα, this study performed an strategy to fabricate 17α-estradiol (E2) probe using gold nanoparticles (AuNP) in breast cancer cells. The positive AuNP probe contain E2-derivated incubate whole cell lysate, and negative AuNP probes were functionalized with polyethylene glycol (PEG) without E2-derivated. Applied this approach reducing the nonspecific binding of E2-derivated related to ERα, at the same time this approach following liquid chromatography tandem mass spectrometry to identify ERα related proteins. The indirect E2-derivated approach pulldown might give a new view on chemical proteomics approach in ERα related protein by LC-MS/MS and present an easy and repeatable experiment.

參考文獻
1. Manning, G.; Whyte, D. B.; Martinez, R.; Hunter, T.; Sudarsanam, S., The protein kinase complement of the human genome. Science 2002, 298 (5600), 1912-+.
2. Wasinger, V. C.; Cordwell, S. J.; Cerpapoljak, A.; Yan, J. X.; Gooley, A. A.; Wilkins, M. R.; Duncan, M. W.; Harris, R.; Williams, K. L.; Humpherysmith, I., Progress with Gene-Product Mapping of the Mollicutes - Mycoplasma-Genitalium. Electrophoresis 1995, 16 (7), 1090-1094.
3. HumpherySmith, I.; Cordwell, S. J.; Blackstock, W. P., Proteome research: Complementarity and limitations with respect to the RNA and DNA worlds. Electrophoresis 1997, 18 (8), 1217-1242.
4. (a) Rix, U.; Superti-Furga, G., Target profiling of small molecules by chemical proteomics. Nature chemical biology 2009, 5 (9), 616-24; (b) Yates, J. R.; Ruse, C. I.; Nakorchevsky, A., Proteomics by Mass Spectrometry: Approaches, Advances, and Applications. Annu Rev Biomed Eng 2009, 11, 49-79.
5. Hartley, B. S., Strategy and Tactics in Protein Chemistry - First Bdh Lecture. Biochemical Journal 1970, 119 (5), 805-&.
6. (a) Berggard, T.; Linse, S.; James, P., Methods for the detection and analysis of protein-protein interactions. Proteomics 2007, 7 (16), 2833-2842; (b) Phizicky, E. M.; Fields, S., Protein-Protein Interactions - Methods for Detection and Analysis. Microbiol Rev 1995, 59 (1), 94-123.
7. Nibbe, R. K.; Markowitz, S.; Myeroff, L.; Ewing, R.; Chance, M. R., Discovery and Scoring of Protein Interaction Subnetworks Discriminative of Late Stage Human Colon Cancer. Molecular & Cellular Proteomics 2009, 8 (4), 827-845.
8. Cheng, P. C.; Chang, H. K.; Chen, S. H., Quantitative nanoproteomics for protein complexes (QNanoPX) related to estrogen transcriptional action. Molecular & cellular proteomics : MCP 2010, 9 (2), 209-24.
9. Moon, Y. W.; Park, S.; Sohn, J. H.; Kang, D. R.; Koo, J. S.; Park, H. S.; Chung, H. C.; Park, B. W., Clinical significance of progesterone receptor and HER2 status in estrogen receptor-positive, operable breast cancer with adjuvant tamoxifen. J Cancer Res Clin 2011, 137 (7), 1123-1130.
10. Puig, O.; Caspary, F.; Rigaut, G.; Rutz, B.; Bouveret, E.; Bragado-Nilsson, E.; Wilm, M.; Seraphin, B., The tandem affinity purification (TAP) method: a general procedure of protein complex purification. Methods 2001, 24 (3), 218-29.
11. (a) Wulfkuhle, J. D.; McLean, K. C.; Paweletz, C. P.; Sgroi, D. C.; Trock, B. J.; Steeg, P. S.; Petricoin, E. F., New approaches to proteomic analysis of breast cancer. Proteomics 2001, 1 (10), 1205-1215; (b) Wolters, D. A.; Washburn, M. P.; Yates, J. R., An automated multidimensional protein identification technology for shotgun proteomics. Analytical chemistry 2001, 73 (23), 5683-5690; (c) Henzel, W. J.; Billeci, T. M.; Stults, J. T.; Wong, S. C.; Grimley, C.; Watanabe, C., Identifying proteins from two-dimensional gels by molecular mass searching of peptide fragments in protein sequence databases. Proceedings of the National Academy of Sciences of the United States of America 1993, 90 (11), 5011-5; (d) Wilkins, M. R.; Sanchez, J. C.; Williams, K. L.; Hochstrasser, D. F., Current challenges and future applications for protein maps and post-translational vector maps in proteome projects. Electrophoresis 1996, 17 (5), 830-838.
12. Edman, P., A method for the determination of amino acid sequence in peptides. Archives of biochemistry 1949, 22 (3), 475.
13. Yassin, A. F.; Haggenei, B.; Budzikiewicz, H.; Schaal, K. P., Fatty-Acid and Polar Lipid-Composition of the Genus Amycolatopsis - Application of Fast-Atom-Bombardment Mass-Spectrometry to Structure-Analysis of Underivatized Phospholipids. Int J Syst Bacteriol 1993, 43 (3), 414-420.
14. (a) Loo, J. A.; Quinn, J. P.; Ryu, S. I.; Henry, K. D.; Senko, M. W.; McLafferty, F. W., High-resolution tandem mass spectrometry of large biomolecules. Proceedings of the National Academy of Sciences of the United States of America 1992, 89 (1), 286-9; (b) Whitehouse, C. M.; Dreyer, R. N.; Yamashita, M.; Fenn, J. B., Electrospray interface for liquid chromatographs and mass spectrometers. Analytical chemistry 1985, 57 (3), 675-9.
15. Berkenkamp, S.; Kirpekar, F.; Hillenkamp, F., Infrared MALDI mass spectrometry of large nucleic acids. Science 1998, 281 (5374), 260-262.
16. Yates, J. R., Mass spectrometry and the age of the proteome. Journal of Mass Spectrometry 1998, 33 (1), 1-19.
17. Wilm, M.; Mann, M., Analytical properties of the nanoelectrospray ion source. Analytical chemistry 1996, 68 (1), 1-8.
18. Washburn, M. P.; Wolters, D.; Yates, J. R., Large-scale analysis of the yeast proteome by multidimensional protein identification technology. Nature biotechnology 2001, 19 (3), 242-247.
19. de la Mora, J. F.; Van Berkel, G. J.; Enke, C. G.; Cole, R. B.; Martinez-Sanchez, M.; Fenn, J. B., Electrochemical processes in electrospray ionization mass spectrometry - Discussion. Journal of Mass Spectrometry 2000, 35 (8), 939-952.
20. Jensen, O. N.; Podtelejnikov, A. V.; Mann, M., Identification of the components of simple protein mixtures by high accuracy peptide mass mapping and database searching. Analytical chemistry 1997, 69 (23), 4741-4750.
21. Shevchenko, A.; Sunyaev, S.; Loboda, A.; Shevehenko, A.; Bork, P.; Ens, W.; Standing, K. G., Charting the proteomes of organisms with unsequenced genomes by MALDI-quadrupole time of flight mass spectrometry and BLAST homology searching. Analytical chemistry 2001, 73 (9), 1917-1926.
22. Gstaiger, M.; Aebersold, R., Applying mass spectrometry-based proteomics to genetics, genomics and network biology. Nat Rev Genet 2009, 10 (9), 617-627.
23. Domon, B.; Aebersold, R., Review - Mass spectrometry and protein analysis. Science 2006, 312 (5771), 212-217.
24. Ambrosino, C.; Tarallo, R.; Bamundo, A.; Cuomo, D.; Franci, G.; Nassa, G.; Paris, O.; Ravo, M.; Giovane, A.; Zambrano, N.; Lepikhova, T.; Janne, O. A.; Baumann, M.; Nyman, T. A.; Cicatiello, L.; Weisz, A., Identification of a Hormone-regulated Dynamic Nuclear Actin Network Associated with Estrogen Receptor alpha in Human Breast Cancer Cell Nuclei. Molecular & Cellular Proteomics 2010, 9 (6), 1352-1367.
25. Mann, M.; Jensen, O. N., Proteomic analysis of post-translational modifications. Nature biotechnology 2003, 21 (3), 255-261.
26. (a) Brill, L. M.; Salomon, A. R.; Ficarro, S. B.; Mukherji, M.; Stettler-Gill, M.; Peters, E. C., Robust phosphoproteomic profiling of tyrosine phosphorylation sites from human T cells using immobilized metal affinity chromatography and tandem mass spectrometry. Analytical chemistry 2004, 76 (10), 2763-2772; (b) Wu, C. J.; Hsu, J. L.; Huang, S. Y.; Chen, S. H., Mapping N-Terminus Phosphorylation Sites and Quantitation by Stable Isotope Dimethyl Labeling. J Am Soc Mass Spectr 2010, 21 (3), 460-471.
27. Wiese, S.; Reidegeld, K. A.; Meyer, H. E.; Warscheid, B., Protein labeling by iTRAQ: A new tool for quantitative mass spectrometry in proteome research. Proteomics 2007, 7 (3), 340-350.
28. (a) Hsu, J. L.; Huang, S. Y.; Chow, N. H.; Chen, S. H., Stable-isotope dimethyl labeling for quantitative proteomics. Analytical chemistry 2003, 75 (24), 6843-6852; (b) Huang, S. Y.; Tsai, M. L.; Wu, C. J.; Hsu, J. L.; Ho, S. H.; Chen, S. H., Quantitation of protein phosphorylation in pregnant rat uteri using stable isotope dimethyl labeling coupled with IMAC. Proteomics 2006, 6 (6), 1722-1734.
29. Henzel, W. J.; Watanabe, C.; Stults, J. T., Protein identification: The origins of peptide mass fingerprinting. J Am Soc Mass Spectr 2003, 14 (9), 931-942.
30. Bronchud, M. H., Principles of molecular oncology. 3rd ed.; Humana Press: Totowa, N.J., 2008; p xix, 418 p.
31. Martucci, C. P.; Fishman, J., P450 Enzymes of Estrogen Metabolism. Pharmacol Therapeut 1993, 57 (2-3), 237-257.
32. Fukuzawa, K.; Mochizuki, Y.; Tanaka, S.; Kitaura, K.; Nakano, T., Molecular interactions between estrogen receptor and its ligand studied by the ab initio fragment molecular orbital method. J Phys Chem B 2006, 110 (32), 16102-16110.
33. Anstead, G. M.; Carlson, K. E.; Katzenellenbogen, J. A., The estradiol pharmacophore: ligand structure-estrogen receptor binding affinity relationships and a model for the receptor binding site. Steroids 1997, 62 (3), 268-303.
34. Tarallo, R.; Bamundo, A.; Nassa, G.; Nola, E.; Paris, O.; Ambrosino, C.; Facchiano, A.; Baumann, M.; Nyman, T. A.; Weisz, A., Identification of proteins associated with ligand-activated estrogen receptor alpha in human breast cancer cell nuclei by tandem affinity purification and nano LC-MS/MS. Proteomics 2011, 11 (1), 172-179.
35. Baron, R.; Willner, B.; Willner, I., Biomolecule-nanoparticle hybrids as functional units for nanobiotechnology. Chem Commun (Camb) 2007, (4), 323-32.
36. (a) Katayama, H.; Oda, Y., Chemical proteomics for drug discovery based on compound-immobilized affinity chromatography. Journal of chromatography. B, Analytical technologies in the biomedical and life sciences 2007, 855 (1), 21-7; (b) Lacerda, S. H. D.; Park, J. J.; Meuse, C.; Pristinski, D.; Becker, M. L.; Karim, A.; Douglas, J. F., Interaction of Gold Nanoparticles with Common Human Blood Proteins. Acs Nano 2010, 4 (1), 365-379.
37. Bakri, S. J.; Pulido, J. S.; Mukherjee, P.; Marler, R. J.; Mukhopadhyay, D., Absence of histologic retinal toxicity of intravitreal nanogold in a rabbit model. Retina-J Ret Vit Dis 2008, 28 (1), 147-149.
38. Aubin-Tam, M. E.; Hamad-Schifferli, K., Structure and function of nanoparticle-protein conjugates. Biomed Mater 2008, 3 (3).
39. (a) Wilchek, M.; Bayer, E. A., Introduction to Avidin-Biotin Technology. Method Enzymol 1990, 184, 5-13; (b) Wang, R. E.; Hunt, C. R.; Chen, J. W.; Taylor, J. S., Biotinylated quercetin as an intrinsic photoaffinity proteomics probe for the identification of quercetin target proteins. Bioorganic & medicinal chemistry 2011, 19 (16), 4710-4720.
40. (a) Lu, Y.; Bottari, P.; Turecek, F.; Aebersold, R.; Gelb, M. H., Absolute quantification of specific proteins in complex mixtures using visible isotope-coded affinity tags. Analytical chemistry 2004, 76 (14), 4104-4111; (b) Xu, N. S.; Yang, H. M.; Cui, M.; Wan, C. H.; Liu, S. Y., High-Performance Liquid Chromatography-Electrospray Ionization-Mass Spectrometry Ligand Fishing Assay: A Method for Screening Triplex DNA Binders from Natural Plant Extracts. Analytical chemistry 2012, 84 (5), 2562-2568.