白細胞介素2 (interleukin-2, IL-2)是受到活化T細胞所產生的重要細胞激素，透過與其受體結合調控著細胞傳遞訊息的功能。具有高親合力的白細胞介素2受體是分別由α (CD25)、β、和γ三條長胜肽鏈所組成，曾有文獻推測白細胞介素2辨識其受體的關鍵組成是特定序列的胜肽鏈與多甘露糖(high mannose)鏈。本論文主要以親合質譜術來研究白細胞介素2與其受體結合的作用。在論文的第一部分是利用分子生物技術來大量表達重組白細胞介素2 (rIL-2)作為探針。將卵白蛋白(ovalbumin)上的多甘露糖(Man-5-Asn)以共價鍵結裝配於磁性奈米粒子表面作為探針，可分離白細胞介素2且直接以基質輔助雷射脫附游離飛行式質譜儀(MALDI-TOF MS)分析，由質譜圖可證明Man-5-Asn能專一地分離出白細胞介素2，此結果也再次確認多甘露糖在白細胞介素2辨識其受體中扮演重要的角色。接著在尋找與Man-5-Asn結合的決定位(epitope)實驗中，我們發現白細胞介素2的胺基酸序列中79到94可能參與Man-5-Asn的辨識及結合，此實驗結果也與最近文獻中X-光繞射所得到結構具有一致性，將更進一步的利用競爭實驗和醣晶片技術去確認決定位的正確性。我們證明了利用奈米探針結合質譜分析可有效的研究醣和蛋白質之間的作用，並可快速鑑定醣和蛋白質的結合位置。
最後實驗部分是將重組白細胞介素2與奈米粒子共價鍵結(MNP@rIL-2)作為探針，從CTLL-2細胞株分離出受體蛋白CD25。利用離心分離技術從CTLL-2細胞株取得膜蛋白且將膜蛋白與MNP@rIL-2反應，當CD25與重組白細胞介素2反應後，利用磁鐵可分離奈米粒子複合體且將其以西方點墨法分析之;雖然經過MNP@rIL-2萃取後並沒有成功的偵測到CD25的訊號，我們推測失敗的因素可能有下列兩點:第一，合成MNP@rIL-2的過程中需要更穩定rIL-2的生物活性。第二，分離膜蛋白的過程中視否改變了白細胞介素2受體固有的生物結構。如何維持白細胞介素2與其受體上的醣之間微弱親合力將是未來實驗修正的方向。

Interleukin-2 (IL-2) is an important cytokine synthesized by activated T cell and well known as a mediator of cellular signalling through interaction with its receptor, IL-2R. The high affinity IL-2R is a heterotrimer composed of α, β, and γ-polypeptide chains. Specific peptide sequence and high mannose-type oligosaccharides on IL-2Rα (CD25) play a critical role in the IL-2 recognition to IL-2R.
In this thesis, we aim to develop new methodology, nanoprobe-based affinity mass spectrometry (NBAMS), to study the interaction between IL-2 and its receptor. In the first part of the thesis, rIL-2 was successfully expressed in E. coli to serve as a probe protein. Oligomannose-5-Asn glycan (Man-5-Asn) prepared from ovalbumin was covalently conjugated on magnetic nanoparticle (MNP@Man-5-Asn) to simultaneously isolate rIL-2 for the analysis of MALDI-TOF MS without an additional elution step. The clean spectrum showed the specific and efficient isolation of rIL-2 by MNP@Man-5-Asn. The results confirmed the importance of oligomannose for rIL-2 recognition by its receptor. Subsequent mapping the binding sequence of Man-5-Asn-interacting epitope revealed that peptide H79-L94 in rIL-2 potentially participates in the rIL-2 recognition of Man-5-Asn. Our results were consistent with the complex structure of IL-2—receptor that was recently determined by X-ray crystallography. In order for further validation, competition assay and carbohydrate array technology were also performed on few synthetic peptide sequences. We demonstrated the combination of functionalized nanoprobe and mass spectrometry provides efficient analysis for probing the carbohydrate-protein interaction and mapping the epitope sequences.
In addition, rIL-2 encapsulated nanoparticles (MNP@rIL-2) was used to as a probe to isolate CD25 from CTLL-2 cell. Membrane fraction from CTLL-2 cells was prepared by centrifugation and incubated with MNP@rIL-2. After glycoprotein-protein interaction, nanoparticles/ bound protein complexes were isolated by magnet and analyzed by Western blotting. So far, we failed to detect CD25 signal after MNP@rIL-2 enrichment. We speculate two possibilities of failure of detection. First, the biological activity of rIL-2 on MNP@rIL-2 may require further optimization by specific immobilization of IL-2 with active conformation. Secondly, the extraction process of membrane fraction may change the native structure of CD25 and/or the β and γ-polypeptide chains. How to maintain the weak IL-2 —oligomannose receptor interaction remain future study.

1. Brandhuber, B. J., Boone, T., Kenney, W. C., and McKay, D. B. (1987) Science 238(4834), 1707-1709
2. Taniguchi, T., Matsui, H., Fujita, T., Takaoka, C., Kashima, N., Yoshimoto, R., and Hamuro, J. (1983) Nature 302(5906), 305-310
3. Smith, K. A. (1988) Science 240(4856), 1169-1176
4. Jain, J., Loh, C., and Rao, A. (1995) Curr Opin Immunol 7(3), 333-342
5. Katsiari, C. G., and Tsokos, G. C. (2006) Autoimmun Rev 5(2), 118-121
6. Williams, T. M., Montoya, G., Wu, Y., Eddy, R. L., Byers, M. G., and Shows, T. B. (1992) Genomics 14(1), 194-196
7. Leonard, W. J., Depper, J. M., Robb, R. J., Waldmann, T. A., and Greene, W. C. (1983) Proc Natl Acad Sci U S A 80(22), 6957-6961
8. Balkwill, F. R. (1989) cytokines in cancer therapy
9. Grabstein, K., Dower, S., Gillis, S., Urdal, D., and Larsen, A. (1986) J Immunol 136(12), 4503-4508
10. Robb, R. J., Greene, W. C., and Rusk, C. M. (1984) J Exp Med 160(4), 1126-1146
11. Hatakeyama, M., Minamoto, S., Uchiyama, T., Hardy, R. R., Yamada, G., and Taniguchi, T. (1985) Nature 318(6045), 467-470
12. Leonard, W. J., Depper, J. M., Crabtree, G. R., Rudikoff, S., Pumphrey, J., Robb, R. J., Kronke, M., Svetlik, P. B., Peffer, N. J., Waldmann, T. A., and et al. (1984) Nature 311(5987), 626-631
13. Nikaido, T., Shimizu, A., Ishida, N., Sabe, H., Teshigawara, K., Maeda, M., Uchiyama, T., Yodoi, J., and Honjo, T. (1984) Nature 311(5987), 631-635
14. Cosman, D., Cerretti, D. P., Larsen, A., Park, L., March, C., Dower, S., Gillis, S., and Urdal, D. (1984) Nature 312(5996), 768-771
15. Hatakeyama, M., Tsudo, M., Minamoto, S., Kono, T., Doi, T., Miyata, T., Miyasaka, M., and Taniguchi, T. (1989) Science 244(4904), 551-556
16. Takeshita, T., Asao, H., Ohtani, K., Ishii, N., Kumaki, S., Tanaka, N., Munakata, H., Nakamura, M., and Sugamura, K. (1992) Science 257(5068), 379-382
17. Chiang, S. S. W. (2005) Th1/Th2 imbalance in schizophrenia: An immunological exploration of etiology
18. Robb, R. J., and Greene, W. C. (1983) J Exp Med 158(4), 1332-1337
19. Robb, R. J., Rusk, C. M., Yodoi, J., and Greene, W. C. (1987) Proc Natl Acad Sci U S A 84(7), 2002-2006
20. Minami, Y., Kono, T., Miyazaki, T., and Taniguchi, T. (1993) Annu Rev Immunol 11, 245-268
21. Robb, R. J., Rusk, C. M., and Neeper, M. P. (1988) Proc Natl Acad Sci U S A 85(15), 5654-5658
22. Takeshita, T., Asao, H., Suzuki, J., and Sugamura, K. (1990) Int Immunol 2(5), 477-480
23. Kondo, M., Takeshita, T., Ishii, N., Nakamura, M., Watanabe, S., Arai, K., and Sugamura, K. (1993) Science 262(5141), 1874-1877
24. Anderson, D. M., Kumaki, S., Ahdieh, M., Bertles, J., Tometsko, M., Loomis, A., Giri, J., Copeland, N. G., Gilbert, D. J., Jenkins, N. A., and et al. (1995) J Biol Chem 270(50), 29862-29869
25. Arima, N., Kamio, M., Imada, K., Hori, T., Hattori, T., Tsudo, M., Okuma, M., and Uchiyama, T. (1992) J Exp Med 176(5), 1265-1272
26. Fukushima, K., Hara-Kuge, S., Ideo, H., and Yamashita, K. (2001) J Biol Chem 276(33), 31202-31208
27. Greene, W. C., and Leonard, W. J. (1986) Annu Rev Immunol 4, 69-95
28. Miyajima, A., Kitamura, T., Harada, N., Yokota, T., and Arai, K. (1992) Annu Rev Immunol 10, 295-331
29. Quan, W. D., Jr., Walker, P. R., Quan, F. M., Ramirez, M., Elsamaloty, H. M., Ghai, V., Vinogradov, M., and Liles, D. K. (2006) Cancer Biother Radiopharm 21(5), 437-442
30. Quan, W. D., Jr., and Quan, F. M. (2003) Cancer Biother Radiopharm 18(4), 535-538
31. Mazza, P., Bocchia, M., Tumietto, F., Costigliola, P., Coronado, O., Bandini, G., Conte, R., Ricchi, E., Vianelli, N., Raise, E., and et al. (1992) Eur J Haematol 49(1), 1-6
32. Rosenberg, S. A., Lotze, M. T., Yang, J. C., Aebersold, P. M., Linehan, W. M., Seipp, C. A., and White, D. E. (1989) Ann Surg 210(4), 474-484; discussion 484-475
33. Nelson, B. H. (2004) J Immunol 172(7), 3983-3988
34. Barber, M. R., and Yang, T. J. (1999) Anticancer Res 19(2A), 1137-1142
35. Dwek, R. A. (1996) Chem Rev 96(2), 683-720
36. Sherblom, A. P., Sathyamoorthy, N., Decker, J. M., and Muchmore, A. V. (1989) J Immunol 143(3), 939-944
37. Zanetta, J. P., Alonso, C., and Michalski, J. C. (1996) Biochem J 318 ( Pt 1), 49-53
38. Tai, T., Yamashita, K., Ogata-Arakawa, M., Koide, N., Muramatsu, T., Iwashita, S., Inoue, Y., and Kobata, A. (1975) J Biol Chem 250(21), 8569-8575
39. Ito, S., Yamashita, K., Spiro, R. G., and Kobata, A. (1977) J Biochem (Tokyo) 81(6), 1621-1631
40. Liang, C. J., Yamashita, K., and Kobata, A. (1980) J Biochem (Tokyo) 88(1), 51-58
41. Kornfeld, R., and Kornfeld, S. (1985) Annu Rev Biochem 54, 631-664
42. Helenius, A., and Aebi, M. (2001) Science 291(5512), 2364-2369
43. Greene, W. C., Depper, J. M., Kronke, M., and Leonard, W. J. (1985) J Cell Sci Suppl 3, 97-106
44. Ofek, I. (1995) Methods Enzymol 253, 528-536
45. R. Michael Lawrence1, S. A. B., Olga M. Fryszman3, Michael A. Poss3. (1997), 553-558
46. Hay, O. M. R. W. a. F. C. (2001) Epitope mapping
47. Kundhavai Natchiar, S., Arockia Jeyaprakash, A., Ramya, T. N., Thomas, C. J., Suguna, K., Surolia, A., and Vijayan, M. (2004) Acta Crystallogr D Biol Crystallogr 60(Pt 2), 211-219
48. Neumann, D., Lehr, C. M., Lenhof, H. P., and Kohlbacher, O. (2004) Adv Drug Deliv Rev 56(4), 437-457
49. Wand, A. J. (2001) Nat Struct Biol 8(11), 926-931
50. M. Karas, D. B., U. Bahr and F. Hillenkamp. (1987) International Journal of Mass Spectrometry and Ion Processes 78, 53-68
51. Mary Gimon-Kinsel, L. M. P.-S., Gary R. Kinsel, and David H. Russell. (1997) J. Am. Chem. Soc. 119(10)
52. Land, C. M., and Kinsel, G. R. (2001) J Am Soc Mass Spectrom 12(6), 726-731
53. Damon I. Papac, J. H., and Kenneth B. Tomer. (1994) 66, 2609 - 2613
54. Nelson, R. W., Krone, J. R., Bieber, A. L., and Williams, P. (1995) Anal Chem 67(7), 1153-1158
55. Brockman, A. H., and Orlando, R. (1995) Anal Chem 67(24), 4581-4585
56. Bundy, J., and Fenselau, C. (1999) Anal Chem 71(7), 1460-1463
57. Tseng, K., Wang, H., Lebrilla, C. B., Bonnell, B., and Hedrick, J. (2001) Anal Chem 73(15), 3556-3561
58. Cao, Y. C., Jin, R., and Mirkin, C. A. (2002) Science 297(5586), 1536-1540
59. Nam, J. M., Thaxton, C. S., and Mirkin, C. A. (2003) Science 301(5641), 1884-1886
60. Bucak, S., Jones, D. A., Laibinis, P. E., and Hatton, T. A. (2003) Biotechnol Prog 19(2), 477-484
61. Gu, H., Ho, P. L., Tsang, K. W., Wang, L., and Xu, B. (2003) J Am Chem Soc 125(51), 15702-15703
62. Gu, H., Ho, P. L., Tsang, K. W., Yu, C. W., and Xu, B. (2003) Chem Commun (Camb) (15), 1966-1967
63. Lin, Y. S., Tsai, P. J., Weng, M. F., and Chen, Y. C. (2005) Anal Chem 77(6), 1753-1760
64. Chou, P. H., Chen, S. H., Liao, H. K., Lin, P. C., Her, G. R., Lai, A. C., Chen, J. H., Lin, C. C., and Chen, Y. J. (2005) Anal Chem 77(18), 5990-5997
65. Po-Chiao Lin, P.-H. C., Shu-Hua Chen, Hsin-Kai Liao, Kai-Yi Wang, Yu-Ju Chen, Dr., Chun-Cheng Lin, Dr. (2006) small 2, 485-489
66. Kriz, K., Ibraimi, F., Lu, M., Hansson, L. O., and Kriz, D. (2005) Anal Chem 77(18), 5920-5924
67. Kong, X. L., Huang, L. C., Hsu, C. M., Chen, W. H., Han, C. C., and Chang, H. C. (2005) Anal Chem 77(1), 259-265
68. Xu, H., Aylott, J. W., Kopelman, R., Miller, T. J., and Philbert, M. A. (2001) Anal Chem 73(17), 4124-4133
69. Chen, C. T., and Chen, Y. C. (2005) Anal Chem 77(18), 5912-5919
70. Cioci, G., Mitchell, E. P., Gautier, C., Wimmerova, M., Sudakevitz, D., Perez, S., Gilboa-Garber, N., and Imberty, A. (2003) FEBS Lett 555(2), 297-301
71. Sanders, D. A., Moothoo, D. N., Raftery, J., Howard, A. J., Helliwell, J. R., and Naismith, J. H. (2001) J Mol Biol 310(4), 875-884
72. Lu, X., and Zhu, H. (2005) Mol Cell Proteomics 4(12), 1948-1958
73. Chen, Y. J., Chen, S. H., Chien, Y. Y., Chang, Y. W., Liao, H. K., Chang, C. Y., Jan, M. D., Wang, K. T., and Lin, C. C. (2005) Chembiochem 6(7), 1169-1173
74. Barre, A., Bourne, Y., Van Damme, E. J., Peumans, W. J., and Rouge, P. (2001) Biochimie 83(7), 645-651
75. Rickert, M., Wang, X., Boulanger, M. J., Goriatcheva, N., and Garcia, K. C. (2005) Science 308(5727), 1477-1480
76. Stauber, D. J., Debler, E. W., Horton, P. A., Smith, K. A., and Wilson, I. A. (2006) Proc Natl Acad Sci U S A 103(8), 2788-2793
77. Wang, X., Rickert, M., and Garcia, K. C. (2005) Science 310(5751), 1159-1163