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通过 MALDI-TOF-MS/MS 进行蓖麻毒素样本的提取和分析的新方法。

A New Method for Extraction and Analysis of Ricin Samples through MALDI-TOF-MS/MS.

机构信息

Brazilian Army CBRN Defense Institute (IDQBRN), Avenida das Américas 28705, Rio de Janeiro 23020-470, Brazil.

Army Institute of Biology (IBEx), Rua Francisco Manuel, 102, Rio de Janeiro 20911-270, Brazil.

出版信息

Toxins (Basel). 2019 Apr 3;11(4):201. doi: 10.3390/toxins11040201.

DOI:10.3390/toxins11040201
PMID:30987210
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC6520692/
Abstract

We report for the first time the efficient use of accelerated solvent extraction (ASE) for extraction of ricin to analytical purposes, followed by the combined use of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), and MALDI-TOF MS/MS method. That has provided a fast and unambiguous method of ricin identification for in real cases of forensic investigation of suspected samples. Additionally, MALDI-TOF MS was applied to characterize the presence and the toxic activity of ricin in irradiated samples. Samples containing ricin were subjected to ASE, irradiated with different dosages of gamma radiation, and analyzed by MALDI-TOF MS/MS for verification of the intact protein signal. For identification purposes, samples were previously subjected to SDS-PAGE, for purification and separation of the chains, followed by digestion with trypsin, and analysis by MALDI-TOF MS/MS. The results were confirmed by verification of the amino acid sequences of some selected peptides by MALDI-TOF MS/MS. The samples residual toxic activity was evaluated through incubation with a DNA substrate, to simulate the attack by ricin, followed by MALDI-TOF MS/MS analyses.

摘要

我们首次报道了加速溶剂萃取（ASE）在分析目的物蓖麻毒素提取中的有效应用，随后结合使用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）、基质辅助激光解吸/电离飞行时间质谱（MALDI-TOF MS）和 MALDI-TOF MS/MS 方法。这为实际的法医调查可疑样本中蓖麻毒素的鉴定提供了一种快速而明确的方法。此外，MALDI-TOF MS 还用于表征辐照样品中蓖麻毒素的存在和毒性活性。含有蓖麻毒素的样品经过 ASE 提取，用不同剂量的γ射线辐照，并通过 MALDI-TOF MS/MS 进行分析，以验证完整蛋白信号的存在。为了进行鉴定，样品首先经过 SDS-PAGE，进行纯化和分离链，然后用胰蛋白酶消化，并通过 MALDI-TOF MS/MS 进行分析。通过 MALDI-TOF MS/MS 对一些选定肽段的氨基酸序列进行验证，对结果进行了确认。通过与 DNA 底物孵育来评估样品的残留毒性活性，以模拟蓖麻毒素的攻击，然后进行 MALDI-TOF MS/MS 分析。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5f53/6520692/679dfa8cc9bc/toxins-11-00201-g009.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5f53/6520692/0c9570fc4aad/toxins-11-00201-g001.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5f53/6520692/ef9329b3c2ad/toxins-11-00201-g002.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5f53/6520692/13a9b1f9674d/toxins-11-00201-g003.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5f53/6520692/86ce9626f0a7/toxins-11-00201-g004.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5f53/6520692/6785eae44e06/toxins-11-00201-g005.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5f53/6520692/6c9f2555d2c4/toxins-11-00201-g006.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5f53/6520692/9a6b35d9113c/toxins-11-00201-g007.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5f53/6520692/d8175b4e2b07/toxins-11-00201-g008.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5f53/6520692/679dfa8cc9bc/toxins-11-00201-g009.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5f53/6520692/0c9570fc4aad/toxins-11-00201-g001.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5f53/6520692/ef9329b3c2ad/toxins-11-00201-g002.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5f53/6520692/13a9b1f9674d/toxins-11-00201-g003.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5f53/6520692/86ce9626f0a7/toxins-11-00201-g004.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5f53/6520692/6785eae44e06/toxins-11-00201-g005.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5f53/6520692/6c9f2555d2c4/toxins-11-00201-g006.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5f53/6520692/9a6b35d9113c/toxins-11-00201-g007.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5f53/6520692/d8175b4e2b07/toxins-11-00201-g008.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5f53/6520692/679dfa8cc9bc/toxins-11-00201-g009.jpg

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