High-performance liquid chromatography-mass selective detection assay for adenine released from a synthetic RNA substrate by ricin A chain
High-performance liquid chromatography-mass selective detection assay for adenine released from a synthetic RNA substrate by ricin A chain
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DOI：
10.1016/j.ab.2004.03.046
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发表时间：
2004-07-01
影响因子：
2.9
通讯作者：
Hale, ML
中科院分区：
文献类型：
--
作者：
Hines, HB;Brueggemann, EE;Hale, ML
采用高效液相色谱法（HPLC）和选择离子监测质谱法（MS）建立了一种定量测定蓖麻毒素A链从合成RNA底物中释放的腺嘌呤的方法。由于蓖麻毒蛋白和蓖麻毒蛋白A链在生物治疗剂和生物武器中具有潜在的应用，因此需要进行测定来评估效力和潜在的活性抑制剂。腺嘌呤的检测限为0.02 μ M（2.4 ng/ml），标准曲线在27.3 μ M以下呈线性。定量下限为0.27 μ M，在此范围内可重复。反应表征表明，大多数腺嘌呤在5小时内释放，并且在甲酸浓度高达0.75 mM（通常用于HPLC-MS的最大值）时，反应不能完全停止。每隔2分钟进样一次，在反冲洗色谱柱前可进样10次，在色谱柱流出液中未观察到蓖麻毒素A链。由于蓖麻毒素A链不通过HPLC柱，因此该测定也可用于蓖麻毒素。通过对该系统的微小修改，该测定应提供对大多数核糖体失活蛋白的活性的快速、灵敏、选择性和定量评估。此外，进一步的色谱和质谱改进可以减少样品要求和分析时间。(C)2004爱思唯尔公司All rights reserved.
High-performance liquid chromatography (HPLC) and selected ion monitoring mass spectrometry (MS) were used to develop a quantitative assay for adenine released from a synthetic RNA substrate by ricin A chain, which contains the toxin's N-glycosidase activity. Because ricin and ricin A chain have potential applications Lis biotherapeutics and bioweapons, assays are needed to evaluate potency and potential inhibitors of activity. The detection limit for adenine was 0.02 muM (2.4 ng/ml), and the standard curve was linear up to 27.3 muM. The lower limit of quantitation was 0.27 muM and was reproducible throughout this range. Reaction characterization showed that most adenine was released by 5 h and that the reaction could not be fully stopped with formic acid concentrations up to 0.75 mM (the maximum typically used for HPLC-MS). Injections were made at 2-min intervals, 10 injections Could be performed before the column was backflushed, and no ricin A chain was observed in the column effluent. This assay would also be useful for ricin since ricin A chain did not pass through the HPLC column. With minor modifications to this system, the assay should provide rapid, sensitive, selective, and quantitative assessment of the activitity of most ribosome-inactivating proteins. In addition, further chromatographic and mass spectrometric improvements Could reduce sample requirements and analysis times. (C) 2004 Elsevier Inc. All rights reserved.