Improved Sensitivity for the Qualitative and Quantitative Analysis of Active Ricin by MALDI-TOF Mass Spectrometry.
Improved Sensitivity for the Qualitative and Quantitative Analysis of Active Ricin by MALDI-TOF Mass Spectrometry.
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DOI：
10.1021/acs.analchem.6b01486
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发表时间：
2016-07-05
影响因子：
7.4
通讯作者：
Kalb SR
中科院分区：
文献类型：
--
作者：
Wang D;Baudys J;Barr JR;Kalb SR
蓖麻毒素是一种高毒性蛋白质，通过阻断蛋白质合成导致细胞死亡，被认为是一种潜在的生物恐怖剂。作为公共卫生和反生物恐怖主义反应的紧急要求，需要快速和灵敏地检测各种类型的样品基质中的蓖麻毒素。通过对底物、反应条件和样品制备的优化，改进了基于MALDI TOF MS的蓖麻毒素脱嘌呤活性体外检测方法。在该方法中，蓖麻毒素被特异性多克隆抗体捕获，然后进行水解反应。蓖麻毒素活性通过检测合成低聚物底物的独特裂解产物来测定。通过使用更有效的RNA底物和优化缓冲液组分、pH和反应温度，增强了对脱嘌呤底物的检测。此外，还研究了MALDI基质、样品制备和样品分离等因素对质谱分析的影响，以提高脱嘌呤产物的电离度和分析的重现性。与优化的参数，0.2 ng/mL的蓖麻毒素加标在缓冲液和牛奶的检测限完成，代表两个以上的数量级的增强，测定灵敏度。提高测定的耐用性或可重复性也使得有可能定量检测具有三个数量级动态范围的活性蓖麻毒素。
Ricin is a highly toxic protein which causes cell death by blocking protein synthesis and is considered a potential bioterrorism agent. Rapid and sensitive detection of ricin toxin in various types of sample matrices is needed as an emergency requirement for public health and anti-bioterrorism response. An in vitro MALDI TOF MS-based activity assay that detects ricin mediated depurination of synthetic substrate was improved through optimization of the substrate, reaction conditions and sample preparation. In this method, the ricin is captured by an specific polycolonal antibody followed by hydrolysis reaction. The ricin activity is determined by detecting the unique cleavage product of synthetic oligomer substrates. The detection of depurinated substrate was enhanced by using a more efficient RNA substrate and optimizing buffer components, pH and reaction temperature. In addition, the factors involved in mass spectrometry analysis, such as MALDI matrix, plate and sample preparation, were also investigated to improve the ionization of the depurinated product and assay reproducibility. With optimized parameters, the limit of detection of 0.2 ng/mL of ricin spiked in buffer and milk was accomplished, representing more than two orders of magnitude enhancement in assay sensitivity. Improving assay’s ruggeddness or reporducibility also made it possible to quantitatively detect active ricin with a three order of magnitude dynamic range.
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影响因子：
14.9
作者：
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通讯作者：
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通讯作者：
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