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作者翁国锋（2019）在《基于质谱技术的寡核苷酸分离分析方法研究》一文中研究指出：寡核苷酸作为一类序列较短的核酸分子,是生命科学和生物医学研究中调节基因表达的基本工具。人工合成的寡核苷酸通过化学修饰可以增加其在生物体内的稳定性,并被开发为基因治疗靶向药物,用于许多与基因相关的疾病如病毒、肿瘤和遗传病等的治疗。与此同时,如何快速、有效、准确地鉴别寡核苷酸所携带的修饰类型以及修饰位点也为其分离分析方法的开发带来了挑战。核酸和蛋白质是两类密切相关的生物大分子,通过彼此间的相互作用可以对各自的生命周期和生物学功能进行调节。核酸蛋白间的相互作用是决定细胞内平衡的关键因素,破坏核酸蛋白的相互作用将会导致细胞的功能紊乱以及相关疾病的发生。核酸与蛋白相互作用的研究涉及寡核苷酸与多肽的液相质谱联用分析,而寡核苷酸和多肽分子所存在的理化性质差异使得两者的同时分析难以实现。基于寡核苷酸质谱分析的现实需求和研究难点,包括传统离子对试剂引起的寡核苷酸质谱信号抑制、寡核苷酸在质谱正离子模式灵敏度低、寡核苷酸难以和多肽在正离子模式同时进行液相质谱联用分析以及寡核苷酸化学修饰和序列信息的快速鉴定等,本论文针对性地提出了改进的分析策略,具体内容包括以下三部分:有机气氛辅助的电喷雾离子化策略用于提高寡核苷酸的质谱检测灵敏度:寡核苷酸的液相质谱联用分析主要存在两方面的挑战,分别是由离子对试剂引起的质谱信号抑制以及寡核苷酸在质谱正离子模式下的灵敏度较低。我们通过将高浓度的醇类气氛引入封闭的电喷雾离子源,成功缓解了高浓度离子对试剂(100mM TEAA)所引起的寡核苷酸在质谱负离子模式下的信号抑制效应。在该缓冲体系中,相比于常规离子化方式,寡核苷酸样品dT15在异丙醇气氛的辅助下,其质谱信号强度提高了约600倍。随后,寡核苷酸的高效液相分离和高灵敏度质谱检测得以同时实现。另外,通过丙酸气氛的辅助,寡核苷酸得以在质谱正离子模式下成功检测,且并无明显加合峰出现。相比于直接通过溶液加酸的方式,丙酸气氛辅助下的寡核苷酸质谱正离子模式信号强度提高至少10倍以上。最后,通过丙酸气氛辅助的离子化策略,寡核苷酸和多肽以及组蛋白样品得以同时在正离子模式下进行分析,且相互之间干扰并不明显。总而言之,有机气氛辅助的离子化策略将会对寡核苷酸的生物学功能探索提供有效的帮助。离子对反相色谱与正离子模式质谱联用下的寡核苷酸分离分析:由于自身携带有电负性极强的磷酸二酯骨架结构,寡核苷酸通常通过离子对反相色谱与负离子模式下的电喷雾质谱联用进行分析。我们通过显著提高源内裂解能量,成功缓解了由离子对试剂三乙胺和六氟异丙醇引起的寡核苷酸在质谱正离子模式下的大量三乙胺加合峰干扰。随后,我们将该策略用于RNA型寡核苷酸的酸解测序分析,发现正离子模式下的测序结果较传统负离子模式更具优势。最终,采用该策略,我们成功实现了寡核苷酸样品与多肽样品在正离子模式下的离子对反相色谱质谱联用分析,且相互间的干扰较少。采用不同基质辅助的激光解析质谱源内裂解下的小干扰RNA的修饰位点鉴定及序列表征:人工合成的医用siRNA由于携带大量的化学修饰以及其裂解碎片在电喷雾离子化过程中容易携带多电荷产生信号干扰,通过串联质谱进行序列鉴定其质谱结果通常过于复杂且难以分析。我们通过激光解析质谱的源内裂解方式并配合不同的基质sDHB和3-HPA对多修饰的siRNA样品SR-2M2-AS进行了修饰位点鉴定和序列表征。siRNA源内裂解的主要碎片类型与所用基质密切相关,采用基质sDHB得到d/y型碎片离子而采用基质3-HPA则得到了a/w型碎片离子,而两种基质分别得到的源内裂解结果都可用于siRNA的快速测序。另外,2’-F修饰将会阻止院内裂解碎片的产生,对此我们提出了相应的裂解机理。在掌握了不同基质下的siRNA源内裂解规律后,这必将成为一种快捷、简单、准确和有效的用于siRNA修饰位点鉴定和序列分析的方法。
Abstract
gua he gan suan zuo wei yi lei xu lie jiao duan de he suan fen zi ,shi sheng ming ke xue he sheng wu yi xue yan jiu zhong diao jie ji yin biao da de ji ben gong ju 。ren gong ge cheng de gua he gan suan tong guo hua xue xiu shi ke yi zeng jia ji zai sheng wu ti nei de wen ding xing ,bing bei kai fa wei ji yin zhi liao ba xiang yao wu ,yong yu hu duo yu ji yin xiang guan de ji bing ru bing du 、zhong liu he wei chuan bing deng de zhi liao 。yu ci tong shi ,ru he kuai su 、you xiao 、zhun que de jian bie gua he gan suan suo xie dai de xiu shi lei xing yi ji xiu shi wei dian ye wei ji fen li fen xi fang fa de kai fa dai lai le tiao zhan 。he suan he dan bai zhi shi liang lei mi qie xiang guan de sheng wu da fen zi ,tong guo bi ci jian de xiang hu zuo yong ke yi dui ge zi de sheng ming zhou ji he sheng wu xue gong neng jin hang diao jie 。he suan dan bai jian de xiang hu zuo yong shi jue ding xi bao nei ping heng de guan jian yin su ,po huai he suan dan bai de xiang hu zuo yong jiang hui dao zhi xi bao de gong neng wen luan yi ji xiang guan ji bing de fa sheng 。he suan yu dan bai xiang hu zuo yong de yan jiu she ji gua he gan suan yu duo tai de ye xiang zhi pu lian yong fen xi ,er gua he gan suan he duo tai fen zi suo cun zai de li hua xing zhi cha yi shi de liang zhe de tong shi fen xi nan yi shi xian 。ji yu gua he gan suan zhi pu fen xi de xian shi xu qiu he yan jiu nan dian ,bao gua chuan tong li zi dui shi ji yin qi de gua he gan suan zhi pu xin hao yi zhi 、gua he gan suan zai zhi pu zheng li zi mo shi ling min du di 、gua he gan suan nan yi he duo tai zai zheng li zi mo shi tong shi jin hang ye xiang zhi pu lian yong fen xi yi ji gua he gan suan hua xue xiu shi he xu lie xin xi de kuai su jian ding deng ,ben lun wen zhen dui xing de di chu le gai jin de fen xi ce lve ,ju ti nei rong bao gua yi xia san bu fen :you ji qi fen fu zhu de dian pen wu li zi hua ce lve yong yu di gao gua he gan suan de zhi pu jian ce ling min du :gua he gan suan de ye xiang zhi pu lian yong fen xi zhu yao cun zai liang fang mian de tiao zhan ,fen bie shi you li zi dui shi ji yin qi de zhi pu xin hao yi zhi yi ji gua he gan suan zai zhi pu zheng li zi mo shi xia de ling min du jiao di 。wo men tong guo jiang gao nong du de chun lei qi fen yin ru feng bi de dian pen wu li zi yuan ,cheng gong huan jie le gao nong du li zi dui shi ji (100mM TEAA)suo yin qi de gua he gan suan zai zhi pu fu li zi mo shi xia de xin hao yi zhi xiao ying 。zai gai huan chong ti ji zhong ,xiang bi yu chang gui li zi hua fang shi ,gua he gan suan yang pin dT15zai yi bing chun qi fen de fu zhu xia ,ji zhi pu xin hao jiang du di gao le yao 600bei 。sui hou ,gua he gan suan de gao xiao ye xiang fen li he gao ling min du zhi pu jian ce de yi tong shi shi xian 。ling wai ,tong guo bing suan qi fen de fu zhu ,gua he gan suan de yi zai zhi pu zheng li zi mo shi xia cheng gong jian ce ,ju bing mo ming xian jia ge feng chu xian 。xiang bi yu zhi jie tong guo rong ye jia suan de fang shi ,bing suan qi fen fu zhu xia de gua he gan suan zhi pu zheng li zi mo shi xin hao jiang du di gao zhi shao 10bei yi shang 。zui hou ,tong guo bing suan qi fen fu zhu de li zi hua ce lve ,gua he gan suan he duo tai yi ji zu dan bai yang pin de yi tong shi zai zheng li zi mo shi xia jin hang fen xi ,ju xiang hu zhi jian gan rao bing bu ming xian 。zong er yan zhi ,you ji qi fen fu zhu de li zi hua ce lve jiang hui dui gua he gan suan de sheng wu xue gong neng tan suo di gong you xiao de bang zhu 。li zi dui fan xiang se pu yu zheng li zi mo shi zhi pu lian yong xia de gua he gan suan fen li fen xi :you yu zi shen xie dai you dian fu xing ji jiang de lin suan er zhi gu jia jie gou ,gua he gan suan tong chang tong guo li zi dui fan xiang se pu yu fu li zi mo shi xia de dian pen wu zhi pu lian yong jin hang fen xi 。wo men tong guo xian zhe di gao yuan nei lie jie neng liang ,cheng gong huan jie le you li zi dui shi ji san yi an he liu fu yi bing chun yin qi de gua he gan suan zai zhi pu zheng li zi mo shi xia de da liang san yi an jia ge feng gan rao 。sui hou ,wo men jiang gai ce lve yong yu RNAxing gua he gan suan de suan jie ce xu fen xi ,fa xian zheng li zi mo shi xia de ce xu jie guo jiao chuan tong fu li zi mo shi geng ju you shi 。zui zhong ,cai yong gai ce lve ,wo men cheng gong shi xian le gua he gan suan yang pin yu duo tai yang pin zai zheng li zi mo shi xia de li zi dui fan xiang se pu zhi pu lian yong fen xi ,ju xiang hu jian de gan rao jiao shao 。cai yong bu tong ji zhi fu zhu de ji guang jie xi zhi pu yuan nei lie jie xia de xiao gan rao RNAde xiu shi wei dian jian ding ji xu lie biao zheng :ren gong ge cheng de yi yong siRNAyou yu xie dai da liang de hua xue xiu shi yi ji ji lie jie sui pian zai dian pen wu li zi hua guo cheng zhong rong yi xie dai duo dian he chan sheng xin hao gan rao ,tong guo chuan lian zhi pu jin hang xu lie jian ding ji zhi pu jie guo tong chang guo yu fu za ju nan yi fen xi 。wo men tong guo ji guang jie xi zhi pu de yuan nei lie jie fang shi bing pei ge bu tong de ji zhi sDHBhe 3-HPAdui duo xiu shi de siRNAyang pin SR-2M2-ASjin hang le xiu shi wei dian jian ding he xu lie biao zheng 。siRNAyuan nei lie jie de zhu yao sui pian lei xing yu suo yong ji zhi mi qie xiang guan ,cai yong ji zhi sDHBde dao d/yxing sui pian li zi er cai yong ji zhi 3-HPAze de dao le a/wxing sui pian li zi ,er liang chong ji zhi fen bie de dao de yuan nei lie jie jie guo dou ke yong yu siRNAde kuai su ce xu 。ling wai ,2’-Fxiu shi jiang hui zu zhi yuan nei lie jie sui pian de chan sheng ,dui ci wo men di chu le xiang ying de lie jie ji li 。zai zhang wo le bu tong ji zhi xia de siRNAyuan nei lie jie gui lv hou ,zhe bi jiang cheng wei yi chong kuai jie 、jian chan 、zhun que he you xiao de yong yu siRNAxiu shi wei dian jian ding he xu lie fen xi de fang fa 。
论文参考文献
论文详细介绍
论文作者分别是来自浙江大学的翁国锋，发表于刊物浙江大学2019-07-17论文，是一篇关于寡核苷酸论文,离子对反相色谱论文,质谱论文,气氛辅助离子化论文,源内裂解论文,浙江大学2019-07-17论文的文章。本文可供学术参考使用，各位学者可以免费参考阅读下载，文章观点不代表本站观点，资料来自浙江大学2019-07-17论文网站，若本站收录的文献无意侵犯了您的著作版权，请联系我们删除。
标签：寡核苷酸论文; 离子对反相色谱论文; 质谱论文; 气氛辅助离子化论文; 源内裂解论文; 浙江大学2019-07-17论文;