Direct Acetonitrile-Assisted Trypsin Digestion Method Combined with LC–MS/MS-Targeted Peptide Analysis for Unambiguous Identification of Intact Ricin,Journal of Proteome Research

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Direct Acetonitrile-Assisted Trypsin Digestion Method Combined with LC–MS/MS-Targeted Peptide Analysis for Unambiguous Identification of Intact Ricin
Journal of Proteome Research ( IF 3.8 ) Pub Date : 2020-10-27 , DOI: 10.1021/acs.jproteome.0c00458
Chang-Cai Liu _{1,

2} , Long-Hui Liang ₁ , Yang Yang _{1,

2} , Hui-Lan Yu _{1,

2} , Long Yan _{1,

2} , Xiao-Sen Li _{1,

2} , Bo Chen ₁ , Shi-Lei Liu _{1,

2} , Hai-Ling Xi ₁

Affiliation

Ricin is a type II ribosome-inactivating protein toxin consisting of A and B chains linked by one interchain disulfide bond. Because of its high toxicity depending on both chains together, confirming the presence of both A and B chains of intact ricin is required during the investigation of the illegal production and application. Here, we report a novel and sensitive acetonitrile (ACN)-assisted trypsin digestion method for unambiguous identification of intact ricin by simultaneous detection of its marker peptides from A and B chains. Marker peptides were generated with a simple procedure by direct cleaving the native ricin at 45 °C for 4 h using Promega modified sequencing grade trypsin under the assistance of 10% ACN, and then directly analyzed by ultrahigh performance liquid chromatography tandem mass spectrometry. The type of trypsin was found to be one critical factor for cleavage of intact ricin based on a significant difference in the yields of specific peptides generated while using various types of trypsin. A low content of ACN in enzymatic buffer significantly reduced the digestion time from overnight to 4 h. There was commonly a better MS response of marker peptides when using the developed ACN-assisted trypsin digestion method than methanol-assisted trypsin digestion within the same 4 h. Totally, seven specific peptides with high sensitivity and specificity including three in the A-chain (TA7, TA11, and TA10) and four in the B-chain (TB6, TB14-ss-TB16, TB20, and TB18) were obtained as good marker peptides for unambiguous identification of intact ricin. The lowest concentration of native ricin for unambiguous identification was 20 ng/mL, in which three marker peptides from both the A-chain and B-chain could be measured with a minimum of three ion transitions. Combined with affinity enrichment, the developed approach was successfully applied for the measurement of intact ricin from the complicated matrix samples of the second, third, and fourth biotoxin exercises organized by the Organisation for the Prohibition of Chemical Weapons (OPCW). This study has provided a recommended detection method combined with one novel ACN-assisted trypsin digestion with MS for forensic unambiguous confirmation of trace ricin intact with high confidence.

中文翻译：

直接乙腈辅助胰蛋白酶消化方法结合LC-MS / MS靶向肽分析可明确鉴定完整蓖麻毒素

蓖麻毒素是II型核糖体失活蛋白毒素，由通过一个链间二硫键连接的A和B链组成。由于其依赖于两条链的高毒性，因此在调查非法生产和使用过程中，需要确认完整蓖麻毒素的A链和B链均存在。在这里，我们报告了一种新颖而灵敏的乙腈（ACN）辅助胰蛋白酶消化方法，通过同时从A和B链检测其标记肽来明确鉴定完整的蓖麻毒蛋白。通过在10％ACN的辅助下使用Promega修饰的测序级胰蛋白酶在45°C下直接裂解天然蓖麻毒蛋白4 h，以简单的程序生成标记肽，然后直接通过超高效液相色谱串联质谱法进行分析。基于使用各种类型的胰蛋白酶时产生的特定肽的产量的显着差异，发现胰蛋白酶的类型是切割完整蓖麻毒蛋白的关键因素。酶缓冲液中低含量的ACN可将消化时间从过夜显着减少到4小时。当使用开发的ACN辅助胰蛋白酶消化方法时，通常在相同的4小时内，标记肽的MS反应要好于甲醇辅助胰蛋白酶消化。总共获得了七种具有高灵敏度和特异性的特异性肽，包括在A链中的三个（TA7，TA11和TA10）和在B链中的四个（TB6，TB14-ss-TB16，TB20和TB18）。用于明确鉴定完整蓖麻毒素的标记肽。用于明确鉴定的天然蓖麻毒蛋白的最低浓度为20 ng / mL，其中可以用最少三个离子跃迁测量来自A链和B链的三个标记肽。结合亲和力富集，该开发的方法已成功应用于由化学武器组织（OPCW）组织的第二，第三和第四次生物毒素演习的复杂基质样品中完整蓖麻毒蛋白的测量。这项研究提供了一种推荐的检测方法，结合一种新型的ACN辅助胰蛋白酶和MS消化技术，可以高可信度地法医明确地鉴定完整的痕量蓖麻毒蛋白。这项开发的方法已成功地用于由化学武器组织（OPCW）组织的第二，第三和第四次生物毒素演习的复杂基质样品中完整蓖麻毒蛋白的测定。这项研究提供了一种推荐的检测方法，结合一种新型的ACN辅助胰蛋白酶和MS消化技术，可以高可信度地法医明确地鉴定完整的痕量蓖麻毒蛋白。这项开发的方法已成功地用于由化学武器组织（OPCW）组织的第二，第三和第四次生物毒素演习的复杂基质样品中完整蓖麻毒蛋白的测定。这项研究提供了一种推荐的检测方法，结合一种新型的ACN辅助胰蛋白酶和MS消化技术，可以高可信度地法医明确地鉴定完整的痕量蓖麻毒蛋白。

更新日期：2021-01-01

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