Rapid and sensitive identification of ricin in environmental samples based on lactamyl agarose beads using LC‐MS/MS (MRM)

Abstract

Ricin, a plant-derived toxin extracted from the seeds of Ricinus communis (castor bean plant), is one of the most toxic proteins known. Ricin's high toxicity, widespread availability, and ease of its extraction make it a potential agent for bioterrorist attacks.Most ricin detection methods are based on immunoassays. These methods may suffer from low efficiency in matrices containing interfering substances, or from false positive results due to antibody cross reactivity, with highly homologous proteins. In this study, we have developed a simple, rapid, sensitive, and selective mass spectrometry assay, for the identification of ricin in complex environmental samples. This assay involves three main stages: (a) Ricin affinity capture by commercial lactamyl-agarose (LA) beads. (b) Tryptic digestion. (c) LC-MS/MS (MRM) analysis of tryptic fragments. The assay was validated using 60 diverse environmental samples such as soil, asphalt, and vegetation, taken from various geographic regions. The assay's selectivity was established in the presence of high concentrations of competing lectin interferences.Based on our findings, we have defined strict criteria for unambiguous identification of ricin. Our novel method, which combines affinity capture beads followed by MRM-based analysis, enabled the identification of 1 ppb ricin spiked into complex environmental matrices. This methodology has the potential to be extended for the identification of ricin in body fluids from individuals exposed (deliberately or accidentally) to the toxin, contaminated food or for the detection of the entire family of RIP-II toxins, by applying multiplex format.

Coupling immuno-magnetic capture with LC–MS/MS(MRM) as a sensitive, reliable, and specific assay for SARS-CoV-2 identification from clinical samples

“…Tryptic digestion was conducted as previously described [ 29 , 38 ]. To evaluate clinical matrix effects on SARS-CoV-2 analysis based on LC–MS/MS(MRM) developed method, 50 mM ammonium bicarbonate (herein termed “buffer”) and NP negative swab samples in viral transport medium (VTM) were spiked with increasing concentrations of SARS-CoV-2 (5 × 10 3 , 1 × 10 4 , 2.5 × 10 4 , 5 × 10 4 , and 1 × 10 5 PFU/ml).…”

“…Tryptic digestion was conducted as previously described [ 29 , 38 ]. Briefly, a total volume of 100 µl of the samples (SARS-CoV-2 purified N or S proteins, buffer spiked viruses or clinical NP swabs) was heated for denaturation (95 °C, 10 min.).…”

Specific and Rapid SARS-CoV-2 Identification Based on LC-MS/MS Analysis

“…In order to evaluate the actual efficiency of tryptic digestion process, recombinant SARS-CoV-2 purified S or N proteins were used. A rapid, simple and efficient tryptic digestion process was performed, similar to the procedure used in our study of protein toxins digestion described recently 29 Table 3 (and detailed in Table S1). The intensities obtained for N derived peptides were ~10-fold higher compared to those observed for the same concentrations of S derived peptides.…”

Specific and Rapid SARS-CoV-2 Identification Based on LC-MS/MS Analysis

“…Tryptic digestion was conducted as previously described 29 . In brief, a total volume of 100 µl samples (SARS-CoV-2 purified N or S proteins, buffer spiked viruses or clinical NP swabs) were heated for denaturation (95°C, 5 min.)…”