Forensic Identification of Neat Ricin and of Ricin from Crude Castor Bean Extracts by Mass Spectrometry

Fredriksson, Sten-Åke; Hulst, A.G.; Artursson, Elisabeth; Jong, A.L. de; Nilsson, Carl-Axel; Baar, Ben L. M. van

doi:10.1021/ac048756u

Cited by 105 publications

(91 citation statements)

References 36 publications

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“…Native gel electrophoresis of ricin showed that the ricin sample (C on the right side of Figure 1) had considerable charge heterogeneity, previously shown by a number of techniques (10,11). The heterogeneity of ricin is due to differences in the amino acid sequences of the subunits and to differences in glycosylation (reviewed in ref 12). Treatment with either bleach or monochloramine resulted in an even more complex pattern, confirming the large number or potential reactions that change the behavior on native gels.…”

Section: Resultsmentioning

confidence: 74%

Process Monitoring the Inactivation of Ricin and Model Proteins by Disinfectants Using Fluorescence and Biological Activity

Cole

Gaigalas

Almeida

2008

Biotechnology Progress

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It is important to develop rapid and reliable processes to monitor the decontamination of toxins released to the environment. The inactivation of the protein toxin ricin by the disinfectants bleach (sodium hypochlorite) and monochloramine was measured by the effect on mammalian cell cytotoxicity. The effect of the disinfectants on the native fluorescence (due mainly to tryptophan and to a lesser extent tyrosine) of ricin was also measured in parallel. Reactions of the disinfectants resulted in a decrease in the native fluorescence that was measured in real time in a noninvasive manner. We compared the inactivation of two well-characterized model enzymes to the behavior of ricin. The model enzymes studied were lysozyme, a small basic enzyme stabilized with internal disulfide bonds, and heart-muscle-type lactate dehydrogenase (LDH), a large protein composed of four subunits. The biological activities of the model enzymes were measured in parallel with their fluorescence. Gel electrophoresis showed a large number of modifications of the proteins caused by the disinfectants reflected in changes in mobility and the formation of higher-order aggregates. Size-exclusion chromatography showed that the disinfectants did not break down the subunit structure of ricin but instead resulted in an increased size and heterogeneity of the protein. Size-exclusion chromatography of LDH indicated that the subunits were dissociated and that higher-order aggregates were also formed. Bleach caused a rapid inactivation of biological activity correlated with a rapid decrease in the fluorescence. Monochloramine required much higher concentrations for significant effects and the kinetics of the reactions were slow, with half-life values of the decrease on the order of minutes. Each protein showed individual differences in responses to the disinfectants, but there was a consistent correlation between the loss of fluorescence and the decrease in biological activity. These results indicate that the monitoring the fluorescence is a useful process with limitations that can be used to monitor the inactivation of toxins using disinfectants.

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Section: Resultsmentioning

confidence: 74%

Process Monitoring the Inactivation of Ricin and Model Proteins by Disinfectants Using Fluorescence and Biological Activity

Cole

Gaigalas

Almeida

2008

Biotechnology Progress

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“…Crude extracts were prepared in house from various cultivars of Ricinus communis castor beans and Abrus precatorius seeds obtained from a local market in Egypt. 22,32 Poros 20 AL aldehyde activated affinity medium was obtained from Applied Biosystems. Millex HA membrane filters were obtained from Merck Millipore and Nanosep MF GHP centrifugal filters from Pall Corporation.…”

Section: ■ Experimental Sectionmentioning

confidence: 99%

“…Crude extracts of ricin and abrin were prepared as described previously. 22 Briefly, 0.5−1 g of seeds were homogenized in 4 mL of diethyl ether and transferred to a screw-capped polypropylene test tube. Four milliliters of 0.5% acetic acid was then added and the suspension was mixed for 30 s on a vortex mixer.…”

Section: ■ Experimental Sectionmentioning

confidence: 99%

“…Nonspecific isolation of toxins using molecular weight filtration has been used for less complex samples. 22,23 Immunoaffinity-based cleanup and enrichment methods have been developed for the determination of ricin in more complex matrices. 24−26 Notably, a protocol involving immunological cleanup followed by trypsin digestion and MALDI MS analysis of the digested products has been used to determine ricin levels in crude ricin extracts and milk samples.…”

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confidence: 99%

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Identification of RIP-II Toxins by Affinity Enrichment, Enzymatic Digestion and LC-MS

et al. 2014

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Type 2 ribosome-inactivating protein toxins (RIP-II toxins) were enriched and purified prior to enzymatic digestion and LC-MS analysis. The enrichment of the RIP-II family of plant proteins, such as ricin, abrin, viscumin, and volkensin was based on their affinity for galactosyl moieties. A macroporous chromatographic material was modified with a galactose-terminated substituent and packed into miniaturized columns that were used in a chromatographic system to achieve up to 1000-fold toxin enrichment. The galactose affinity of the RIP-II proteins enabled their selective enrichment from water, beverages, and extracts of powder and wipe samples. The enriched fractions were digested with trypsin and RIP-II peptides were identified based on accurate mass LC-MS data. Their identities were unambiguously confirmed by LC-MS/MS product ion scans of peptides unique to each of the toxins. The LC-MS detection limit achieved for ricin target peptides was 10 amol and the corresponding detection limit for the full method was 10 fmol/mL (0.6 ng/mL). The affinity enrichment method was applied to samples from a forensic investigation into a case involving the illegal production of ricin and abrin toxins.T ype 2 ribosome-inactivating proteins (RIP-II toxins) are a class of plant toxins that includes a number of potent chem-bio threat agents, such as ricin (Ricinus communis), abrin (Abrus precatorius), viscumin (Viscum album), modeccin (Adenia digitata), and volkensin (Adenia volkensii). RIP-II toxins are heterodimeric proteins that consist of an enzymatically active N-glycosidase A chain connected to a B chain via a disulfide bond. The B chain is a lectin with carbohydrate binding domains that have an affinity for galactose-terminated surface receptors on eukaryotic cells. Binding promotes the uptake of the toxin into the cell by endocytosis, where a part intriguingly finds its way via the Golgi complex to the endoplasmatic reticulum. From there the A subunit is translocated into the cytosol, where it hydrolyses the Nglycosidic bond between an adenine residue and ribose at a specific position in 28S rRNA and thereby inhibits protein synthesis, eventually causing cell death.

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“…RCA60 and RCA120 can be identified by mass spectrometry (MS). [8][9][10] For this purpose, the toxin and its agglutinin are isolated from the matrix. For tandem-MS methods, the proteins are digested enzymatically.…”

Section: Introductionmentioning

confidence: 99%

A Glyco-chip for the Detection of Ricin by an Automated Chemiluminescence Read-out System

et al. 2013

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The plant toxin ricin is a lectin that binds to D-galactose or lactose moieties by multivalent interactions. In the present work, this avidity was used to develop a novel sandwich glyco-immunoassay using a carbohydrate microarray. For realization, 6-azidohexyl-lactose was immobilized on an alkyne silane surface by Cu(I) catalyzed click chemistry. This procedure is fast, and prevents any nonspecific binding on the microarray surface. Ricin binds via its B-chain to the lactose moieties, and is detected by the biotinylated anti-ricin A-chain. By adding a horseradish peroxidase-labeled streptavidin, a chemiluminescence signal can be generated. This method is described as a sandwich-type glyco-immunoassay. The signal on the glyco-chip can be regenerated for at least 10 measurements. The limit of detection was estimated to be 80 ng mL -1 . The assay was carried out on the automated microarray readout platform MCR 3. In this way, it took 20 min for one measurement, including regeneration of the chip surface.

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Forensic Identification of Neat Ricin and of Ricin from Crude Castor Bean Extracts by Mass Spectrometry

Cited by 105 publications

References 36 publications

Process Monitoring the Inactivation of Ricin and Model Proteins by Disinfectants Using Fluorescence and Biological Activity

Process Monitoring the Inactivation of Ricin and Model Proteins by Disinfectants Using Fluorescence and Biological Activity

Identification of RIP-II Toxins by Affinity Enrichment, Enzymatic Digestion and LC-MS

A Glyco-chip for the Detection of Ricin by an Automated Chemiluminescence Read-out System

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