研究生: |
林家宏 Chia-Hung Lin |
---|---|
論文名稱: |
利用聚精胺酸修飾之奈米鑽石濃縮萃取磺酸化胜肽與肝素 Using Polyarginine-coated Nanodiamonds to Enrich and Extract Sulfopeptides and Heparin |
指導教授: |
林震煌
Lin, Cheng-Huang 張煥正 Chang, Huan-Cheng 吳志哲 Wu, Chih-Che |
學位類別: |
碩士 Master |
系所名稱: |
化學系 Department of Chemistry |
論文出版年: | 2011 |
畢業學年度: | 99 |
語文別: | 中文 |
論文頁數: | 57 |
中文關鍵詞: | 奈米鑽石 、磺酸化 、親和性層析 、基質輔助雷射脫附游離飛行時間質譜儀 、肝素 |
英文關鍵詞: | nanodiamond, sulfonation, affinity chromatography, MALDI-TOF MS, heparin |
論文種類: | 學術論文 |
相關次數: | 點閱:92 下載:10 |
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磺酸化與磷酸化反應是控制細胞內蛋白質功能重要的後轉譯修飾 (Post-Translational Modification,PTM),在醣類結構上也是相當常見。細胞表面的蛋白聚醣 (Proteoglycan) 銜接出不同硫酸化雙醣為單位的糖胺多醣 (Glycosaminoglycans,GAGs),例如肝素或硫酸乙醯肝素 (heparin/heparan sulfate) 影響著多種生物反應,包含抗凝血功能、細胞的生長、調控細胞離子濃度、癌症還有細菌或病毒感染,甚至與遺傳疾病都息息相關。基質輔助雷射脫附游離飛行時間質譜儀 (MALDI-TOF MS)是用來分析蛋白質與醣類結構的工具之一,然而後轉譯修飾的醣類或胜肽在生物中含量甚少,所以利用質譜分析之前,樣品的濃縮與純化則是不可或缺的。本篇利用聚精胺酸修飾之奈米鑽石對磺酸化和磷酸化兩種後轉譯修飾胜肽親和性比較,發現磺酸化胜肽具有優先親和性。利用此技術,也能從高含量蔗糖溶液中選擇性萃取微量肝素雙醣。另外還發現於3-aminoquinoline (3-AQ) 基質中添加氨化物1,1,3,3-tetramethylguanidine (TMG) 不只能夠降低肝素雙醣於質譜分析下的磺酸根裂解,還能增強去質子離子的訊號。此奈米鑽石固相萃取技術結合質譜分析硫酸醣類,在未來將有利於了解細胞膜表面發生之病毒感染。
Sulfonation and phosphorylation are important post-translational modifications (PTMs) of protein function in cells and occur frequently in oligosaccharides. The glycosaminoglycans (GAGs), which are characterized by a variably sulfated repeating disaccharide unit, bind with cell surface proteoglycans. For example, heparin/haparan sulfates influence numerous biological processes which include anticoagulation, cellular physiology, ionic strength regulation, cancer, viral invasion, bacteria invasion and genetic diseases. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is one of the tools for the structure analysis of proteins and oligosaccharides. However, the low abundance and low stoichiometry of post-translationally modified oligosaccharides and peptides in organisms make isolation and concentration of the compound indispensable prior to MS analysis. In this study, we utilize polyarginine-coated nanodiamond as a high affinity nanoprobe for sulfopeptides and phosphopeptides. We compared the affinity of there two post-translationally modified peptides toward the nanoparticle and found preferential adsorption of sulfopeptide in their mixture. With this technique, we are able to selectively extract heparin disaccharides in high abundant sucrose solution. Additionally, we found that adding 1,1,3,3-tetramethylguanidine (TMG) to 3-aminoquanoline (3-AQ) matrix not only reduces the sulfate fragmentation of heparin disaccharides but also enhances the signal of the protonated ions of MALDI-TOF MS analysis. We conclude that MALDI MS combined with this nanodiamond–based solid phase extraction is a useful technique. It can facilitate our understanding of viral invasion through interaction with sulfate saccharides on cell membrane surface in the future.
1. Chang, Y.-C.; Huang, C.-N.; Lin, C.-H.; Chang, H.-C.; Wu, C.-C., Mapping protein cysteine sulfonic acid modifications with specific enrichment and mass spectrometry: An integrated approach to explore the cysteine oxidation. Proteomics 2010, 10 (16), 2961-2971.
2. Kong, X.; Huang, L. C. L.; Liau, S.-C. V.; Han, C.-C.; Chang, H.-C., Polylysine-Coated Diamond Nanocrystals for MALDI-TOF Mass Analysis of DNA Oligonucleotides. Anal. Chem. 2005, 77 (13), 4273-4277.
3. Rhee, S. G.; Chae, H. Z.; Kim, K., Peroxiredoxins: A historical overview and speculative preview of novel mechanisms and emerging concepts in cell signaling. Free Radical Biology and Medicine 2005, 38 (12), 1543-1552.
4. Wood, Z. A.; Schr鐰er, E.; Robin Harris, J.; Poole, L. B., Structure, mechanism and regulation of peroxiredoxins. Trends in Biochemical Sciences 2003, 28 (1), 32-40.
5. Negishi, M.; Pedersen, L. G.; Petrotchenko, E.; Shevtsov, S.; Gorokhov, A.; Kakuta, Y.; Pedersen, L. C., Structure and Function of Sulfotransferases. Archives of Biochemistry and Biophysics 2001, 390 (2), 149-157.
6. Sasisekharan, R.; Shriver, Z.; Venkataraman, G.; Narayanasami, U., Roles of heparan-sulphate glycosaminoglycans in cancer. Nat Rev Cancer 2002, 2 (7), 521-528.
7. http://themedicalbiochemistrypage.org/glycans.html.
8. Randall, D. R.; Colobong, K. E.; Hemmelgarn, H.; Sinclair, G. B.; Hetty, E.; Thomas, A.; Bodamer, O. A.; Volkmar, B.; Fernhoff, P. M.; Casey, R.; Chan, A. K.; Mitchell, G.; Stockler, S.; Melancon, S.; Rupar, T.; Clarke, L. A., Heparin cofactor II-thrombin complex: A biomarker of MPS disease. Molecular Genetics and Metabolism 2008, 94 (4), 456-461.
9. Chung, C.-S.; Hsiao, J.-C.; Chang, Y.-S.; Chang, W., A27L Protein Mediates Vaccinia Virus Interaction with Cell Surface Heparan Sulfate. J. Virol. 1998, 72 (2), 1577-1585.
10. Malavaki, C. J.; Theocharis, A. D.; Lamari, F. N.; Kanakis, I.; Tsegenidis, T.; Tzanakakis, G. N.; Karamanos, N. K., Heparan sulfate: biological significance, tools for biochemical analysis and structural characterization. Biomedical Chromatography 2011, 25 (1-2), 11-20.
11. Yang, B.; Solakyildirim, K.; Chang, Y.; Linhardt, R., Hyphenated techniques for the analysis of heparin and heparan sulfate. Analytical and Bioanalytical Chemistry 2011, 399 (2), 541-557.
12. Chang, C.-K.; Wu, C.-C.; Wang, Y.-S.; Chang, H.-C., Selective Extraction and Enrichment of Multiphosphorylated Peptides Using Polyarginine-Coated Diamond Nanoparticles. Anal. Chem. 2008, 80 (10), 3791-3797.
13. Mikkelsen, S. R.; Cortón, E., Enzymes. John Wiley & Sons, Inc.: 2004; p 16-40.
14. Jones, C. J.; Beni, S.; Limtiaco, J. F. K.; Langeslay, D. J.; Larive, C. K., Heparin Characterization: Challenges and Solutions. Annual Review of Analytical Chemistry 2011, 4 (1), null.
15. Mallis, L. M.; Wang, H. M.; Loganathan, D.; Linhardt, R. J., Sequence analysis of highly sulfated, heparin-derived oligosaccharides using fast atom bombardment mass spectrometry. Anal. Chem. 1989, 61 (13), 1453-1458.
16. Karas, M.; Bachmann, D.; Hillenkamp, F., Influence of the wavelength in high-irradiance ultraviolet laser desorption mass spectrometry of organic molecules. Anal. Chem. 1985, 57 (14), 2935-2939.
17. Schiller, J.; Arnhold, J.; Benard, S.; Reichl, S.; Arnold, K., Cartilage degradation by hyaluronate lyase and chondroitin ABC lyase: a MALDI-TOF mass spectrometric study. Carbohydrate Research 1999, 318 (1-4), 116-122.
18. Juhasz, P.; Biemann, K., Mass spectrometric molecular-weight determination of highly acidic compounds of biological significance via their complexes with basic polypeptides. Proc. Natl. Acad. Sci. U. S. A. 1994, 91 (10), 4333-4337.
19. Currie, G. J.; Yates, J. R., Analysis of oligodeoxynucleotides by negative-ion matrix-assisted laser desorption mass spectrometry. J. Am. Soc. Mass Spectrom. 1993, 4 (12), 955-963.
20. Asara, J. M.; Allison, J., Enhanced detection of phosphopeptides in matrix-assisted laser desorption/ionization mass spectrometry using ammonium salts. J. Am. Soc. Mass Spectrom. 1999, 10 (1), 35-44.
21. Tomoya Kinumi, Y. S., Ryuichi Arakawa, Yoshiro Tatsu, Yasushi Shigeri, Noboru Yumoto, Etsuo Niki,, Effective detection of peptides containing cysteine sulfonic acid using matrix-assisted laser desorption/ionization and laser desorption/ionization on porous silicon mass spectrometry. Journal of Mass Spectrometry 2006, 41 (1), 103-112.
22. Naggar, E.; Costello, C.; Zaia, J., Competing fragmentation processes in tandem mass spectra of heparin-like glycosaminoglycans. J. Am. Soc. Mass Spectrom. 2004, 15 (11), 1534-1544.
23. Zaia, J., Principles of mass spectrometry of glycosaminoglycans. J. Biomol. Mass Spectrom. 2005, 1 (1), 3-36.
24. Przybylski, C.; Gonnet, F.; Bonnaffé, D.; Hersant, Y.; Lortat-Jacob, H.; Daniel, R., HABA-based ionic liquid matrices for UV-MALDI-MS analysis of heparin and heparan sulfate oligosaccharides. Glycobiology 2010, 20 (2), 224-234.
25. Fukuyama, Y.; Nakaya, S.; Yamazaki, Y.; Tanaka, K., Ionic Liquid Matrixes Optimized for MALDI-MS of Sulfated/Sialylated/Neutral Oligosaccharides and Glycopeptides. Anal. Chem. 2008, 80 (6), 2171-2179.
26. Fang, D.-C.; Fabian, P.; Szekely, Z.; Fu, X.-Y.; Tang, T.-H.; Csizmadia, I. G., Structure and stability of ammonium-sulfate and guanidium-sulfate complex. Journal of Molecular Structure: THEOCHEM 1998, 430, 161-170.
27. Schug, K. A.; Lindner, W., Noncovalent Binding between Guanidinium and Anionic Groups: Focus on Biological- and Synthetic-Based Arginine/Guanidinium Interactions with Phosph[on]ate and Sulf[on]ate Residues. Chemical Reviews 2004, 105 (1), 67-114.
28. Chen, W.-H.; Lee, S.-C.; Sabu, S.; Fang, H.-C.; Chung, S.-C.; Han, C.-C.; Chang, H.-C., Solid-Phase Extraction and Elution on Diamond (SPEED): A Fast and General Platform for Proteome Analysis with Mass Spectrometry. Anal. Chem. 2006, 78 (12), 4228-4234.
29. Krueger, A., New Carbon Materials: Biological Applications of Functionalized Nanodiamond Materials. Chemistry – A European Journal 2008, 14 (5), 1382-1390.
30. Liu, W. K.; Adnan, A.; Kopacz, A. M.; Hallikainen, M.; Ho, D.; Lam, R.; Lee, J.; Belytschko, T.; Schatz, G.; Tzeng, Y.; Kim, Y.-J.; Baik, S.; Kim, M. K.; Kim, T.; Lee, J.; Hwang, E.-S.; Im, S.; Ōsawa, E.; Barnard, A.; Chang, H.-C.; Chang, C.-C.; Oñate, E., Design of Nanodiamond Based Drug Delivery Patch for Cancer Therapeutics and Imaging Applications. In Nanodiamonds, Ho, D., Ed. Springer US: 2010; pp 249-284.
31. Downard, K. M.; Morrissey, B.; Schwahn, A. B., Mass spectrometry analysis of the influenza virus. Mass Spectrometry Reviews 2009, 28 (1), 35-49.
32. Kruger, A.; Liang, Y.; Jarre, G.; Stegk, J., Surface functionalisation of detonation diamond suitable for biological applications. Journal of Materials Chemistry 2006, 16 (24), 2322-2328.
33. Strott, C. A., Sulfonation and Molecular Action. Endocr. Rev. 2002, 23 (5), 703-732.