Liquid chromatography electrospray ionization mass spectrometry analysis of the ocular metabolites from a short interfering RNA duplex
Liquid chromatography electrospray ionization mass spectrometry analysis of the ocular metabolites from a short interfering RNA duplex
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DOI：
10.1016/j.jchromb.2006.03.008
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发表时间：
2006-05-01
影响因子：
3
通讯作者：
Lockridge, J
中科院分区：
文献类型：
--
作者：
Beverly, M;Hartsough, K;Lockridge, J
通过离子对反相液相色谱（IP-RP-LC）结合电喷雾电离质谱（ESI-MS）检查siRNA双链体（SIRNA-027）的眼部代谢。将RNA双链体眼内注射到新西兰白色兔的眼中。在不同时间点处死兔，并通过IP-RP-LC-MS分析玻璃体和视网膜/脉络膜组织的siRNA。该方法使用具有甲醇梯度的六氟异丙醇（HFIP）/三乙胺（TEA）离子配对缓冲液。使用电喷雾电离，双链体保存在气相中，用于通过三重四极杆质谱仪进行分析。用这种方法鉴定了来自兔眼玻璃体液和视网膜/脉络膜组织的代谢物，并建立了siRNA降解模式。结果表明，双链体主要从一端代谢。计算siRNA双链体的该末端具有两个末端中最弱的结合能，表明siRNA分裂成单链的能力是其降解的一个因素。(c)2006 Elsevier B. V.保留所有权利。
The ocular metabolism of an siRNA duplex, SIRNA-027, was examined by ion-pair reversed-phase liquid chromatography (IP-RP-LC) coupled to electrospray ionization mass spectrometry (ESI-MS). The RNA duplex was injected intraocularly into the eyes of New Zealand white rabbits. Rabbits were sacrificed at different timepoints and the vitreous and retina/choroid tissue analyzed for siRNA by IP-RP-LC-MS. The method used a hexafluoroisopropanol (HFIP)/triethylamine (TEA) ion-pairing buffer with a methanol gradient. Using electrospray ionization, the duplex was preserved in the gas phase for analysis by a triple quadrupole mass spectrometer. With this methodology metabolites from rabbit ocular vitreous humor and retina/choroid tissue were identified and a pattern of siRNA degradation was established. Results showed that the duplex was metabolized predominantly from one end. This end of the siRNA duplex was calculated to have the weakest binding energy of the two ends indicating that the ability of the siRNA to split into single strands is a factor in its degradation. (c) 2006 Elsevier B.V. All rights reserved.