基于巯基-炔基点击化学的苯硼酸功能化材料的制备及其在糖蛋白/糖肽选择性富集中的应用研究
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摘要
硼亲和色谱法在糖肽/糖蛋白选择性富集中的应用趋于成熟。硼酸亲和材料的选择性、生物相容性、制备过程是否简便均是开发新型苯硼酸功能化材料需要考虑的问题。本研究针对硼酸亲和材料开发的关键问题,设计并制备了一种新型苯硼酸功能化材料(TYBA材料)。此材料采用基于巯基-炔基的无铜催化点击化学方法进行合成,生物相容性好,制备方法简便。红外光谱表征结果证明材料合成成功。采用基质辅助激光解吸离子化-飞行时间质谱(MALDI-TOF-MS)测试TYBA材料的糖肽富集选择性,结果表明,TYBA材料能够分别从辣根过氧化物酶(HRP)和免疫球蛋白G(Ig G)酶解液中鉴定出11个和8个糖肽;以HRP-牛血清白蛋白(BSA)酶解液(1∶10,n/n)混合物作为研究对象,富集后能够鉴定出8个糖肽信号。采用SDS-PAGE凝胶电泳考察TYBA材料的糖蛋白富集选择性,以HRP、Ig G、核糖核酸酶B(RNase B)作为考察对象,结果表明,TYBA材料对不同类型糖蛋白具有较好的富集选择性。以实际人血清样品为测试对象验证TYBA材料在实际生物样品中的应用价值。结果表明,富集后非糖蛋白得到较大程度去除,糖蛋白得以富集。所制备的材料和方法具有大规模分离处理实际蛋白质样品的应用前景。
The application of boronic acid affinity chromatography in glycoprotein/glycopeptide enrichment tends to be more and more mature. The enrichment selectivity,biocompatibility,and facile operation protocol are key points for efficient enrichment methods. In this work,a novel boronic acid functionalized material( TYBA material) was prepared by using Thiol-yne click chemistry. The TYBA material was proved to be successfully synthesized by infrared( IR) characterization. The glycopeptide/glycoprotein enrichment selectivity of the TYBA material was evaluated. Matrix assisted laser desorption ionization time-of-flight mass spectrometry( MALDI-TOF MS) was applied for evaluation of glycopeptide enrichment selectivity. Taking digest of horseradish peroxidase( HRP) and immunoglobulin G( IgG) as samples,11 and 8 glycopeptides were identified with improved signals after TYBA material enrichment,respectively. High abundant nonglycopeptides were well removed from the eluting fraction. The result indicated high glycopeptide enrichment selectivity of TYBA material. A mixture of HRP and bovine serum albumin( BSA) enzymatic solution( molar ratio 1∶ 10) was used as the sample and 8 glycopeptide signals were identified after enrichment. Sodium dodecyl sulfate polyacrylamide gel electrophoresis( SDS-PAGE) was applied for evaluation d of enrichment selectivity of glycoprotein. The mixture of HRP,IgG,BSA and RNaseB proteins was used as model sample,and the results showed that TYBA material had high glycoprotein enrichment selectivity. The application of TYBA material in the analysis of real biosample was also evaluated by human plasma. The results indicated the TYBA material could be used in large scale glycoprotein analysis.
The application of boronic acid affinity chromatography in glycoprotein/glycopeptide enrichment tends to be more and more mature. The enrichment selectivity,biocompatibility,and facile operation protocol are key points for efficient enrichment methods. In this work,a novel boronic acid functionalized material( TYBA material) was prepared by using Thiol-yne click chemistry. The TYBA material was proved to be successfully synthesized by infrared( IR) characterization. The glycopeptide/glycoprotein enrichment selectivity of the TYBA material was evaluated. Matrix assisted laser desorption ionization time-of-flight mass spectrometry( MALDI-TOF MS) was applied for evaluation of glycopeptide enrichment selectivity. Taking digest of horseradish peroxidase( HRP) and immunoglobulin G( IgG) as samples,11 and 8 glycopeptides were identified with improved signals after TYBA material enrichment,respectively. High abundant nonglycopeptides were well removed from the eluting fraction. The result indicated high glycopeptide enrichment selectivity of TYBA material. A mixture of HRP and bovine serum albumin( BSA) enzymatic solution( molar ratio 1∶ 10) was used as the sample and 8 glycopeptide signals were identified after enrichment. Sodium dodecyl sulfate polyacrylamide gel electrophoresis( SDS-PAGE) was applied for evaluation d of enrichment selectivity of glycoprotein. The mixture of HRP,IgG,BSA and RNaseB proteins was used as model sample,and the results showed that TYBA material had high glycoprotein enrichment selectivity. The application of TYBA material in the analysis of real biosample was also evaluated by human plasma. The results indicated the TYBA material could be used in large scale glycoprotein analysis.
引文
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26 Zhao Y Y,Yu L,Guo Z M,Li X L,Liang X M. Anal. Bioanal. Chem.,2011,399(10):3359-3365
2 Hirabayashi J,Arata Y,Kasai K. Proteomics,2001,1(2):295-303
3 Yang G L,Tan Z Q,Lu W,Guo J,Yu H J,Yu J M,Sun C W,Qi X W,Li Z,Guan F. J. Proteome Res.,2015,14(2):639-653
4 BAO Hui-Min,XIE Li-Qi,LU Hao-Jie. Chinise Journal of Chromatography,2016,34(12):1145-1153包慧敏,谢力琦,陆豪杰.色谱,2016,34(12):1145-1153
5 SUN Xu-Dong,ZHANG Ling-Yi,ZHANG Wei-Bing. Chinise Journal of Chromatography,2017,35(7):696-702孙旭东,张凌怡,张维冰.色谱,2017,35(7):696-702
6 Zhu F F,Clemmer D E,Trinidad J C. Analyst,2017,142:65-74
7 Liu L T,Yu M,Zhang Y,Wang C C,Lu H J. ACS Appl. Mater. Interfaces,2014,6:7823-7832
8 CHEN Cheng,WANG Hong-Xi,KANG Hong-Jian,YAO Yao,QING Guang-Yan,LI Xiu-Ling,LIANG Xin-Miao. Chinese J. Anal. Chem.,2017,45(8):1149-1154陈成,王宏喜,康虹健,姚要,卿光焱,李秀玲,梁鑫淼.分析化学,2017,45(8):1149-1154
9 Wu C,Liang Y,Zhao Q,Qu Y Y,Zhang S,Wu Q,Liang Z,Zhang L H,Zhang Y K. Chemistry,2014,20(28):8737-8743
10 Alvarez-Manilla G,Atwood J,Guo Y,Warren N L,Orlando R,Pierce M. J. Proteome Res.,2006,5(3):701-708
11 LI Tian-Tian,ZHANG Li-Yuan,YU Zhen-Long,MA Xiao-Chi,DENG Sa. Chinese J. Anal. Chem.,2017,45(9):1259-1263李甜甜,张丽媛,于振龙,马骁驰,邓卅.分析化学,2017,45(9):1259-1263
12 Fournier D,Hoogenboom R,Schubert U S. Chem. Soc. Rev.,2007,36:1369-1380
13 Binder W H,Sachsenhofer R. Macromol. Rapid Commun.,2008,29:952-981
14 LIU Ren-Yong,ZHANG Zhong-Ping. Chinese J. Anal. Chem.,2017,45(12):1956-1962刘仁勇,张忠平.分析化学,2017,45(12):1956-1962
15 Hein J E,Fokin V V. Cheminform,2010,41(28):1302-1315
16 Konkolewicz D,Gray-Weale A,Perrier S. J. Am. Chem. Soc.,2009,131(50):18075-18077
17 Fairbanks B D,Scott T F,Kloxin C J,Anseth K S,Bowman C N. Macromolecules,2009,42(42):211-217
18 Zhang S T,He X W,Chen L X,Zhang Y K. New J. Chem.,2014,38,4212-4218
19 YANG Fan,MAO Zhe,HE Xi-Wen,CHEN Lang-Xing,ZHANG Yu-Kui. Chinese Journal of Chromatography,2013,31(6):531-536杨帆,毛劼,何锡文,陈朗星,张玉奎.色谱,2013,31(6):531-536
20 Bie Z J,Chen Y,Li H Y,Wu R H,Liu Z. Anal. Chim. Acta,2014,834:1-8
21 Qu Y Y,Liu J X,Yang K G,Liang Z,Zhang L H,Zhang Y K. Chem. Eur. J.,2012,18:9056-9062
22 Wang H Y,Bie Z J,Lu C C,Liu Z. Chem. Sci.,2013,4:4298-4303
23 Wang S T,Chen D,Ding J,Yuan B F,Feng Y Q. Chem. Eur. J.,2013,19:606-612
24 Guo Z M,Lei A W,Xu Q. Chem. Commun.,2006,43:4512-4514
25 Li H Y,Wang H Y,Liu Y C,Liu Z. Chem. Commun.,2012,48:4115-4117
26 Zhao Y Y,Yu L,Guo Z M,Li X L,Liang X M. Anal. Bioanal. Chem.,2011,399(10):3359-3365