Recommended Mass Spectrometry-Based Strategies to Identify Ricin-Containing Samples

Abstract

Ricin is a protein toxin produced by the castor bean plant (Ricinus communis) together with a related protein known as R. communis agglutinin (RCA120). Mass spectrometric (MS) assays have the capacity to unambiguously identify ricin and to detect ricin’s activity in samples with complex matrices. These qualitative and quantitative assays enable detection and differentiation of ricin from the less toxic RCA120 through determination of the amino acid sequence of the protein in question, and active ricin can be monitored by MS as the release of adenine from the depurination of a nucleic acid substrate. In this work, we describe the application of MS-based methods to detect, differentiate and quantify ricin and RCA120 in nine blinded samples supplied as part of the EQuATox proficiency test. Overall, MS-based assays successfully identified all samples containing ricin or RCA120 with the exception of the sample spiked with the lowest concentration (0.414 ng/mL). In fact, mass spectrometry was the most successful method for differentiation of ricin and RCA120 based on amino acid determination. Mass spectrometric methods were also successful at ranking the functional activities of the samples, successfully yielding semi-quantitative results. These results indicate that MS-based assays are excellent techniques to detect, differentiate, and quantify ricin and RCA120 in complex matrices.

Biological toxins of potential bioterrorism risk: Current status of detection and identification technology

“…Alternatively, laboratories using highly sophisticated MSbased methods targeting both protein identity and functional activity of ricin delivered convincing results. Several of the successful approaches have been published elsewhere in more detail [19,22,27].…”

“…D) Crystal structure of SEB in complex with MHC II and T cell receptor (4C56.pdb [93]) (SEB red ribbon, intrachain disulfide bridge yellow sticks, MHC II yellow ribbon, TCR green ribbon) and EM picture of Staphylococcus aureus producing SE (insert). substrates; the detection limits are mostly in the low ng/mL to high pg/mL-range [21,22]. Alternatively, cell-based cytotoxicity assays display the biological activity of ricin, some of them in real-time or in a high-throughput format requiring functional blockade by antibodies in order to demonstrate specificity [15].…”

“…As to quantification, mostly ELISA and one quantitative MS approach were used in combination with different in-house calibrants [19,22]. Still, it was possible to determine a consensus concentration on the RM offered in the PT -to our knowledge, this material represents the only thoroughly characterized ricin RM with a consensus concentration defined in an international PT [21].…”

“…In the PT, the MS-based approaches referenced in Table 2 proved to have excellent selectivity between the two related lectins, allowing for unambiguous identification of ricin versus RCA120. Among them were protocols applying liquid chromatography-electrospray ionization mass spectrometry (LC-ESI MS) and matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) MS [21,22]. With respect to functional assays, two different approaches were successfully used on the samples provided: an MS-based method detecting the adenine release from an artificial substrate [21,[24][25][26] and an in vitro cytotoxicity assay based on impedance measurement [21,27].…”

Ricin and Ricinus communis in pharmacology and toxicology-from ancient use and “Papyrus Ebers” to modern perspectives and “poisonous plant of the year 2018”

“…In samples with complex matrices, MS assays have been found capable of unambiguously identifying ricin and its activity (Kalb et al 2015). More specifically, Kalb and coworkers reported on the application of MS-based methods for detection, differentiation, and quantitation of ricin and RCA120 in blinded samples, as part of the EQuATox proficiency test (Establishment of Quality Assurance for the Detection of Biological Toxins of Potential Bioterrorism Risk; Kalb et al 2015).…”