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. 2024 Jul 15;37(7):1218-1228.
doi: 10.1021/acs.chemrestox.4c00149. Epub 2024 Jul 4.

Mass Spectrometric Detection and Differentiation of Enzymatically Active Abrin and Ricin Combined with a Novel Affinity Enrichment Technique

Kaitlyn K Drinkard  1 , John R Barr  1 , Suzanne R Kalb  1
Affiliations

Mass Spectrometric Detection and Differentiation of Enzymatically Active Abrin and Ricin Combined with a Novel Affinity Enrichment Technique

Kaitlyn K Drinkard et al. Chem Res Toxicol. .

Abstract

Abrin and ricin are toxic proteins produced by plants. Both proteins are composed of two subunits, an A-chain and a B-chain. The A-chain is responsible for the enzymatic activity, which causes toxicity. The B-chain binds to glycoproteins on the cell surface to direct the A-chain to its target. Both toxins depurinate 28S rRNA, making it impossible to differentiate these toxins based on only their enzymatic activity. We developed an analytical workflow for both ricin and abrin using a single method and sample. We have developed a novel affinity enrichment technique based on the ability of the B-chain to bind a glycoprotein, asialofetuin. After the toxin is extracted with asialofetuin-coated magnetic beads, an RNA substrate is added. Then, depurination is detected by a benchtop matrix-assisted laser desorption/ionization time-of-flight (MALDI TOF) mass spectrometer to determine the presence or absence of an active toxin. Next, the beads are subjected to tryptic digest. Toxin fingerprinting is done on a benchtop MALDI-TOF MS. We validated the assay through sensitivity and specificity studies and determined the limit of detection for each toxin as nanogram level for enzymatic activity and μg level for toxin fingerprinting. We examined potential cross-reactivity from proteins that are near neighbors of the toxins and examined potential false results in the presence of white powders.

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Conflict of interest statement

The authors declare no competing financial interest.

Figures

Figure 1
Figure 1
Schematic of the analytical workflow which includes an enzyme activity assay to mimic the biological activity of ricin and abrin and peptide fingerprinting to differentiate these toxins.
Figure 2
Figure 2
Mass spectra depicting the RNA substrate with (A) 200 ng/mL of abrin and (B) 8 ng/mL of abrin. Abrin was extracted with 4-APLP beads. The intact RNA substrate can be observed at m/z 4525 and the depurinated substrate can be observed at m/z 4408.
Figure 3
Figure 3
Binding kinetics of ricin B-chain to asialofetuin.
Figure 4
Figure 4
Mass spectra of tryptic digests of (A) no toxin, (B) ricin, and (C) abrin, with toxin fragments indicated by asterisk.
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