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.

Army Institute of Biology Rua Francisco Manuel 102 Rio de Janeiro 20911 270 Brazil

Brazilian Army CBRN Defense Institute Avenida das Américas 28705 Rio de Janeiro 23020 470 Brazil

Chemical Engineering Department Military Institute of Engineering Praca General Tiburcio 80 Rio de Janeiro 22290 270 Brazil

Department of Chemistry Faculty of Science University of Hradec Kralove Rokitanskeho 62 50003 Hradec Kralové Czech Republic

Laboratory of Molecular Modeling Applied to Chemical and Biological Defense Military Institute of Engineering Praca General Tiburcio 80 Rio de Janeiro 22290 270 Brazil

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