建立了双功能试剂2-[(4-异硫氰基苯基)甲基]-1,4,7,10-四氮杂环十二烷-1,4,7,10-四乙酸(p-SCN-Bn-DOTA)与标准肽段反应的新方法,测定了反应产物与镧系金属Eu(Ⅲ)的螯合效率,探索了其作为靶向探针和蛋白定量标记试剂性能。考察了不同缓冲体系的浓度及pH值、p-SCN-Bn-DOTA量与肽段相对量、反应体系中有机相与水相比例,反应温度与反应时间对偶联效率的影响。实验结果显示:p-SCN-Bn-DOTA量为肽段的32倍,缓冲体系为0.2 mol/L TEAB (pH 8.5),60℃反应60 min,有机相与水相之比为4.4:1(V/V)时标记效率高;在HCl/NaAc缓冲体系(pH 4.0)中60℃反应60 min,金属离子鳌合反应效率高达94%。本研究建立了一种新的基于双功能试剂p-SCN-Bn-DOTA的标记方法,并成功用于RGD肽等的标记。
A novel labeling approach with good compatibility based on 2-(4-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (p-SCN-Bn-DOTA) was established. The labeling conditions of p-SCN-Bn-DOTA with standard peptides were optimized, and the labeling efficiency of macrocyclic complex of p-SCN-Bn-DOTA with lanthanide metal ion Eu(Ⅲ) was also investigated. The labeling process was a two-step reaction. The labeling efficiency was affected by several factors including pH, buffer, reaction time, reaction temperature, and ratio of p-SCN-Bn-DOTA to peptide. The optimized conditions were as followed:the first step of labeling reaction was reacted in 0.2 mol/L triethylene diamine (TEAB) (pH 8.5) buffer for 60 min at 60℃, and the molar ratio of p-SCN-Bn-DOTA with peptide was 32. The second step of rare earth metal chelating was in pH 4.0 HCl/NaAC buffer for 60 min at 60℃. This approach was successfully applied in RGD peptide labeling.
参考文献
| [1] | Lee S, Xie J, Chen X. Chem. Rev., 2010,110(5):3087-3111 |
| [2] | Kerr T J, McLean J A. Chem. Commun., 2010,46(30):5479-5481 |
| [3] | Ang C Y, Tan S Y, Zhao Y. Org. Biomol. Chem., 2014,12(27):4776-4806 |
| [4] | Von Wallbrunn A, H ltke C, Zühlsdorf M, Heindel W, Schäfers M, Bremer C. Eur. J. Nucl. Med. Mol. Imaging, 2007,34(5):745-754 |
| [5] | DI Li-Juan, ZHANG Xu-Chu, ZHANG Chun-Li, WANG Rong-Fu. Labeled Immunoassays and Clinical Medicine, 2007,14(1):58-61 邸丽娟, 张旭初, 张春丽, 王荣福.标记免疫分析与临床,2007,14(1):58-61 |
| [6] | HUANG Yan-Yan, ZHAO Rui. Journal of Instrumental Analysis, 2012,32(9):1184-1190 黄嫣嫣, 赵睿.分析测试学报,2012,32(9):1184-1190 |
| [7] | Shiba K. Chem. Soc. Rev., 2010,39(1):117-126 |
| [8] | Gygi S P, Rist B, Gerber S A, Turecek F, Gelb M H, Aebersold R. Nat. Biotechnol., 1999,17(10):994-999 |
| [9] | Yao X, Freas A, Ramirez J, Demirev P A, Fenselau C. Anal. Chem., 2001,73(13):2836-2842 |
| [10] | Ong S E, Blagoev B, Kratchmarova I, Kristensen D B, Steen H, Pandey A, Mann M. Mol. Cell. Proteom., 2002,1(5):376-386 |
| [11] | Ross P L, Huang Y N, Marchese J N, Williamson B, Parker K, Hattan S, Khainovski N, Pillai S, Dey S, Daniels S, Purkayastha S, Juhasz P, Martin S, Bartlet-Jones M, He F, Jacobson A, Pappin D J. Mol. Cell. Proteom., 2004,3(12):1154-1169 |
| [12] | LI Jia-Bin, ZHOU Lian-Qi, YAN Hui, LI Nan-Nan, HAO Fei-Ran, TIAN Fang, ZHANG Yang-Jun. Chinese Journal of Chromatography, 2014,32(4):361-368 李佳斌, 周廉淇, 颜辉, 李楠楠, 郝斐然, 田芳, 张养军.色谱,2014,32(4):361-368 |
| [13] | Whetstone P A, Butlin N G, Corneillie T M, Meares C F. Bioconjug. Chem., 2004,15(1):3-6 |
| [14] | Ahrends R, Pieper S, Kühn A, Weisshoff H, Hamester M, Lindemann T, Scheler C, Lehmann K, Taubner K, Linscheid MW. Mol. Cell. Proteom., 2007,6(11):1907-1916 |
| [15] | He Y, Esteban-Fernández D, Linscheid M W. Talanta, 2015,134:468-475 |
| [16] | XU Ming, YANG Li-Min, WANG Qiu-Quan. Chinese J. Anal.Chem., 2015,43(9):1265-1271 徐 明, 杨利民, 王秋泉.分析化学,2015,43(9):1265-1271 |
| [17] | Liu H, Zhang Y, Wang J, Zhou C, Cai Y, Qian X. Anal. Chem., 2006,78(18), 6614-6621 |
| [18] | Wang X, Zhang Y J, Wang X Y, Lin H J, Qin W J, Qian X H. Anal. Methods, 2012, (4):1629-1632 |
| [19] | Wang X Y, Wang X, Qin W J, Lin H J, Wei J Y, Zhang Y J, Qian X H. Analyst, 2013,138:5309-5317 |
| [20] | Wang Xin, Development and Application of a Novel Method for Multiplex Proteomic Quantitation Based on Metal Element Labeling Coupled with Mass Spectrometry, Academy of Military Medical Science, Beijing. 2012 王 昕.金属元素标记结合质谱技术的蛋白质组多重定量新方法研究及应用,北京:军事医学科学院, 2012 |
| [21] | Jakubowski N, Waentig L, Hayen H, Venkatachalam A, Bohlen A V, Roosb P H, Andreas M. J. Anal. At. Spectrom., 2008,23:1497-1507 |
| [22] | Cooper M S, Ma M T, Sunassee K, Shaw K P, Williams J D, Paul R L, Donnelly P S, Blower P J. Bioconjug. Chem., 2012,23:1029-1039 |
| [23] | Schlesinger J, Koezle I, Bergmann R, Tamburini S, Bolzati C, Tisato F, Noll B, Klussmann S, Vonhoff S, Wuest F, Pietzsch H J, Steinbach J. Bioconjug. Chem., 2008,19(4):928-939 |
| [24] | Fonge H, Huang H, Scollard D, Reilly RM, Allen C. J. Control Release, 2012,157(3):366-374 |
| [25] | Dong S Y, Zhao Z W, Ma H M. J. Proteome Res., 2006,5(1):26-31 |
| [26] | Whetstone P A, Butlin N G, Corneillie T M, Meares C F. Bioconjug. Chem., 2004,15(1):3-6 |
| [27] | Ahrends R, Pieper S, Kühn A, Weisshoff H, Hamester M, Lindemann T, Scheler C, Lehmann K, Taubner K, Linscheid M W. Mol. Cell. Proteom., 2007,6(11):1907-1916 |
| [28] | Izatt R M, Pawlak K, Bradshaw J S, Bruening R L. Chem. Rev., 1991,91(8):1721-2085 |
| [29] | Antczak C, Jaggi J S, LeFave C V, Curcio M J, McDevitt M R, Scheinberg D A. Bioconjug. Chem., 2006,17(6):1551-1560 |
| [30] | MI Wei, WANG Jing, YING Wan-Tao, CAI Yun, QIAN Xiao-Hong. Chinese J. Anal. Chem., 2010,38(10):1393-1399 米 薇, 王晶, 应万涛, 蔡耘, 钱小红.分析化学,2010,38(10):1393-1399 |
| [31] | ZHANG Ai-Hong, LIN Hong-Jun, HUANG Shun-Xiang, ZHOU Xue-Zhi. Jounral of Chinese Mass Spectometry Society, 2014,35(5):427-437 张爱红, 林虹君, 黄顺祥, 周学志.质谱学报,2014,35(5):427-437 |
- 下载量()
- 访问量()
- 您的评分：
-
10%
-
20%
-
30%
-
40%
-
50%