由於近年來人口數目增加及氣候的變遷，造成全球糧食的不足，於是促成基因改造作物蓬勃發展。雖然基因改造作物可以大幅改善缺糧現況，相對的也具有尚未無法證實的未知憂慮及安全性爭議。因此本實驗運用同重元素相對和絕對定量(iTRAQ)的化學標記方法搭配質譜技術，分析基因改造黃豆(DAS-81419-2)和非基因改造黃豆兩者的蛋白質相對含量變化。iTRAQ標定的胜肽樣品會先經由等電點聚焦分離儀(sIEF)、強陽離子交換層析法(SCX)以及鹼性逆向層析法(bRP)進行第一維分離，以降低樣品複雜度，同時藉由三種不同分餾方法提供互補性、正交性以利鑑定到更多的蛋白質。而後進行奈米級液相層析連接質譜儀分析。在此實驗中，總共鑑定到2648個蛋白質以及8831個不重複胜肽。在三種分餾方法中，鹼性逆向層析法效果最好。相較於僅使用鹼性逆向層析法，增加兩種分餾方法可多鑑定到34%的蛋白質及32%的不重複胜肽。此外使用生物資訊軟體分析在基因改造黃豆和非基因改造黃豆中，分析具有顯著差異蛋白質的生物路徑及功能，發現與核糖體、營養儲藏活性及細胞質等路徑有關。本研究為基因改造黃豆(DAS-81419-2)提供了新的觀點，並且期望此數據有利於基因改造作物未來發展。

Due to the increase of popularity and the change of global climate environment, the concern on global food crisis is growing. This phenomenon has caused the genetically modified organisms (GMOs) thriving in recent years. Although the GMOs can dramatically solve the global food crisis, there are some unknown anxieties and the safety issues which are suspicious to the public now. In this study, isobaric tags for relative and absolute quantitation (iTRAQ) technology was applied for the investigation of the protein profiles in both the genetically modified (GM, DAS-81419-2) and the non-genetically modified (non-GM) soybeans. The iTRAQ labeled peptides were fractionated by solution isoelectric focusing (sIEF), strong cationic exchange chromatography (SCX) and basic reverse phase chromatography (bRP), followed by nano-LC tandem mass spectrometric analysis. Two-dimensional liquid chromatography technique that employed on iTRAQ labeled peptides gave results with excellent complementarity, orthogonality and more protein identifications. A total of 2648 proteins including 8831 unique peptides were identified. Moreover, differentially expressed proteins were selected between the GM and the non-GM soybeans for the bioinformatics analysis. They were found in ribosome, nutrient reservoir activity, cytoplasmic pathway, and so on. We expect that it can be beneficial on its future development for genetically modified crops.

1. Edman, P., Method for determination of the amino acid sequence in peptides. Acta chem. scand, 1950. 4(7): p. 283-293.
2. Ma, B., et al., PEAKS: powerful software for peptide de novo sequencing by tandem mass spectrometry. Rapid communications in mass spectrometry, 2003. 17(20): p. 2337-2342.
3. Henzel, W.J., et al., Identifying proteins from two-dimensional gels by molecular mass searching of peptide fragments in protein sequence databases. Proceedings of the National Academy of Sciences, 1993. 90(11): p. 5011-5015.
4. Cleveland, D. W., Fischer, S. G., Kirschner, M. W., & Laemmli, U. K. (1977). Peptide mapping by limited proteolysis in sodium dodecyl sulfate and analysis by gel electrophoresis. Journal of Biological Chemistry, 252(3), 1102-1106.
5. Thiede, B., et al., Peptide mass fingerprinting. Methods, 2005. 35(3): p. 237-247.
6. Eng, J.K., A.L. McCormack, and J.R. Yates, An approach to correlate tandem mass spectral data of peptides with amino acid sequences in a protein database. Journal of the American Society for Mass Spectrometry, 1994. 5(11): p. 976-989.
7. ashburn, M.P., D. Wolters, and J.R. Yates, 3rd, Large-scale analysis of the yeast proteome by multidimensional protein identification technology. Nat Biotechnol, 2001. 19(3): p. 242-7.
8. Zhang, X., et al., Multi-dimensional liquid chromatography in proteomics--a review. Anal Chim Acta, 2010. 664(2): p. 101-13.
9. Kopaciewicz, W., et al., Retention model for high-performance ion-exchange chromatography. Journal of Chromatography A, 1983. 266: p. 3-21.
10. Sestak, J., D. Moravcova, and V. Kahle, Instrument platforms for nano liquid chromatography. J Chromatogr A, 2015. 1421: p. 2-17.
11. Peng, J., et al., Evaluation of multidimensional chromatography coupled with tandem mass spectrometry (LC/LC-MS/MS) for large-scale protein analysis: the yeast proteome. J Proteome Res, 2003. 2(1): p. 43-50.
12. Dowell, J.A., et al., Comparison of two-dimensional fractionation techniques for shotgun proteomics. Anal Chem, 2008. 80(17): p. 6715-23.
13. Lau, E., et al., Combinatorial use of offline SCX and online RP-RP liquid chromatography for iTRAQ-based quantitative proteomics applications. Mol Biosyst, 2011. 7(5): p. 1399-408.
14. Hubner, N.C., S. Ren, and M. Mann, Peptide separation with immobilized pI strips is an attractive alternative to in-gel protein digestion for proteome analysis. PROTEOMICS, 2008. 8(23-24): p. 4862-4872.
15. Horth, P., et al., Efficient fractionation and improved protein identification by peptide OFFGEL electrophoresis. Mol Cell Proteomics, 2006. 5(10): p. 1968-74.
16. Yang, F., et al., High-pH reversed-phase chromatography with fraction concatenation for 2D proteomic analysis. Expert Rev Proteomics, 2012. 9(2): p. 129-34.
17. Spicer, V., et al., 3D HPLC-MS with Reversed-Phase Separation Functionality in All Three Dimensions for Large-Scale Bottom-Up Proteomics and Peptide Retention Data Collection. Anal Chem, 2016. 88(5): p. 2847-55.
18. Kirkland, J.J., M.A. van Straten, and H.A. Claessens, High pH mobile phase effects on silica-based reversed-phase high-performance liquid chromatographic columns. Journal of Chromatography A, 1995. 691(1): p. 3-19.
19. Märk, T.D. and G.H. Dunn, Electron impact ionization. 2013: Springer Science & Business Media.
20. Harrison, A.G., Chemical ionization mass spectrometry. 1992: CRC press.
21. Kaufmann, R., Matrix-assisted laser desorption ionization (MALDI) mass spectrometry: a novel analytical tool in molecular biology and biotechnology. Journal of biotechnology, 1995. 41(2-3): p. 155-175.
22. Fenn, J.B., et al., Electrospray ionization for mass spectrometry of large biomolecules. Science, 1989. 246(4926): p. 64-71.
23. Wilm, M.S. and M. Mann, Electrospray and Taylor-Cone theory, Dole's beam of macromolecules at last? International Journal of Mass Spectrometry and Ion Processes, 1994. 136(2-3): p. 167-180.
24. Kaltashov, I.A. and R.R. Abzalimov, Do ionic charges in ESI MS provide useful information on macromolecular structure? Journal of the American Society for Mass Spectrometry, 2008. 19(9): p. 1239-1246.
25. Schwartz, J.C., M.W. Senko, and J.E. Syka, A two-dimensional quadrupole ion trap mass spectrometer. Journal of the American Society for Mass Spectrometry, 2002. 13(6): p. 659-669.
26. Hager, J.W., A new linear ion trap mass spectrometer. Rapid Communications in Mass Spectrometry, 2002. 16(6): p. 512-526.
27. Wells, J.M. and S.A. McLuckey, Collision‐induced dissociation (CID) of peptides and proteins. Methods in enzymology, 2005. 402: p. 148-185.
28. Wu, W.W., et al., Discovery-and target-based protein quantification using iTRAQ and pulsed Q collision induced dissociation (PQD). Journal of proteomics, 2012. 75(8): p. 2480-2487.
29. Wasinger, V.C., et al., Progress with gene‐product mapping of the Mollicutes: Mycoplasma genitalium. Electrophoresis, 1995. 16(1): p. 1090-1094.
30. Anderson, N.L. and N.G. Anderson, Proteome and proteomics: new technologies, new concepts, and new words. Electrophoresis, 1998. 19(11): p. 1853-1861.
31. Fenselau, C. and X. Yao, Proteolytic Labeling With 18 O for Comparative Proteomics Studies: Preparation of 18 O-Labeled Peptides and the 18 O/16 O Peptide Mixture. Quantitative Proteomics by Mass Spectrometry, 2007: p. 135-142.
32. Heller, M., et al., Trypsin catalyzed 16 O-to-18 O exchange for comparative proteomics: tandem mass spectrometry comparison using MALDI-TOF, ESI-QTOF, and ESI-ion trap mass spectrometers. Journal of the American Society for Mass Spectrometry, 2003. 14(7): p. 704-718.
33. Gygi, S.P., et al., Quantitative analysis of complex protein mixtures using isotope-coded affinity tags. Nature biotechnology, 1999. 17(10): p. 994-999.
34. Thompson, A., et al., Tandem mass tags: a novel quantification strategy for comparative analysis of complex protein mixtures by MS/MS. Analytical chemistry, 2003. 75(8): p. 1895-1904.
35. Evans, C., et al., An insight into iTRAQ: where do we stand now? Analytical and Bioanalytical Chemistry, 2012. 404(4): p. 1011-1027.
36. Wolfenbarger, L. L., & Phifer, P. R. (2000). The ecological risks and benefits of genetically engineered plants. Science, 290(5499), 2088-2093.
37. 袁秋英；林季昌；葉茂生；蔣慕琰 Crop, Enviroment & Bioinformatics 2009, 5, 268-280
38. 牛惠之；郭華仁 基因改造議題-從紛爭到展望；行政院農委會動植物防疫檢疫局, 2004
39. Ocaña, M. F., Fraser, P. D., Patel, R. K., Halket, J. M., & Bramley, P. M. (2009). Evaluation of stable isotope labelling strategies for the quantitation of CP4 EPSPS in genetically modified soya. Analytica chimica acta, 634(1), 75-82.
40. Qin, J., Gu, F., Liu, D., Yin, C., Zhao, S., Chen, H., ... & Zhang, M. (2013). Proteomic analysis of elite soybean Jidou17 and its parents using iTRAQ-based quantitative approaches. Proteome science, 11(1), 12.
41. Li, J., Ding, X., Han, S., He, T., Zhang, H., Yang, L., ... & Gai, J. (2016). Differential proteomics analysis to identify proteins and pathways associated with male sterility of soybean using iTRAQ-based strategy. Journal of proteomics, 138, 72-82.
42. Casey, T.M., et al., Analysis of Reproducibility of Proteome Coverage and Quantitation Using Isobaric Mass Tags (iTRAQ and TMT). Journal of Proteome Research, 2017. 16(2): p. 384-392.
43. Pichler, P., et al., Peptide labeling with isobaric tags yields higher identification rates using iTRAQ 4-plex compared to TMT 6-plex and iTRAQ 8-plex on LTQ Orbitrap. Analytical chemistry, 2010. 82(15): p. 6549-6558.
44. 劉欣. (2008). 大豆球蛋白 glycinin 和 β–conglycinin 引發 Balb/c 小鼠過敏反應及其肌理的研究 (Doctoral dissertation, 浙江: 浙江大學).
45. Wang, T., Qin, G. X., Sun, Z. W., & Zhao, Y. (2014). Advances of research on glycinin and β-conglycinin: a review of two major soybean allergenic proteins. Critical reviews in food science and nutrition, 54(7), 850-862.
46. Wang, T., Qin, G. X., Sun, Z. W., & Zhao, Y. (2014). Advances of research on glycinin and β-conglycinin: a review of two major soybean allergenic proteins. Critical reviews in food science and nutrition, 54(7), 850-862.
47. 羅玉嬌, 李濱, 舒衡平, & 蔣立平. (2012). Kunitz 型胰蛋白酶抑製劑的研究進展 (Doctoral dissertation).
48. Messina, M., & Barnes, S. (1991). The role of soy products in reducing risk of cancer. J Natl Cancer Inst, 83(8), 541-6.
49. Volarević, S., Stewart, M. J., Ledermann, B., Zilberman, F., Terracciano, L., Montini, E., ... & Thomas, G. (2000). Proliferation, but not growth, blocked by conditional deletion of 40S ribosomal protein S6. Science, 288(5473), 2045-2047.
50. Meyuhas, O. (2008). Physiological roles of ribosomal protein S6: one of its kind. International review of cell and molecular biology, 268, 1-37.