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

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.

Development and Evaluation of an Immuno-MALDI-TOF Mass Spectrometry Approach for Quantification of the Abrin Toxin in Complex Food Matrices

“…In the final assay conditions, the LOD was determined at ~40 ng/mL in milk or apple juice. The sensitivity of the new protocol is in line with previous immuno-MALDI-TOF MS methods reported for ricin identification [ 16 , 17 , 25 ], considering that ricin has minimum lethal doses similar to abrin [ 6 , 24 ]. Of note, a time consuming ZipTip separation was necessary in those ricin assays, to purify and concentrate ricin before the MALDI-TOF MS analysis.…”

“…As an alternative, MALDI-time-of-flight (TOF) MS has some advantages over ESI-MS, notably the speed of analysis and the simplicity, with the absence of a prior chromatographic separation. Nevertheless, only few MALDI-TOF MS assays have been described for ricin [ 12 , 15 , 16 , 17 ], and none for abrin.…”

Introduction to the Toxins Special Issue “Ricin Toxins”

“…They confirm these findings by gene expression analysis and show activation of immune regulation pathways and immune cell recruitment after ricin exposure [12]. Sousa et al [13] describe an accelerated solvent extraction (ASE) method followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and MALDI-TOF-MS/MS for extraction and detection of ricin in forensic samples. This method could also detect ricin in gamma-irradiated samples [13].…”

“…Sousa et al [13] describe an accelerated solvent extraction (ASE) method followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and MALDI-TOF-MS/MS for extraction and detection of ricin in forensic samples. This method could also detect ricin in gamma-irradiated samples [13].…”

Theoretical Investigation of Repurposed Drugs Potentially Capable of Binding to the Catalytic Site and the Secondary Binding Pocket of Subunit A of Ricin

“…Also known as castor oil plant, R. communis is the source of the ricin oil, a raw material extensively used in the production of lubricants, green fuel, drugs, and cosmetics. 1 However, the high toxicity of ricin occasionally causes accidental intoxication of workers involved in the oil extraction. Also, the castor oil cake, a byproduct of the ricin oil extraction, is rich in minerals and other nutrients, being, for this reason, used as a fertilizer and to feed livestock.…”