Integrated proteomic and functional mass spectrometry workflow for comprehensive ricin characterization at trace levels.

The plant toxin ricin, derived from the castor bean plant Ricinus communis, is a major bioterrorism agent that requires analytical strategies to confirm both toxin identity and functionality. Ricin consists of an enzymatically active A chain (RTA) and a galactose-binding B chain (RTB) linked by an inter-chain disulfide bond. Conventional bottom-up mass spectrometry identifies ricin using proteotypic peptides from the individual chains but does not establish whether the toxin is present as an intact, active holotoxin. Here, we present an integrated workflow that enables structural and functional detection of ricin present at trace levels in complex matrices. The proteomic mass spectrometry assay combines targeted detection of multiple peptides from both chains with the challenging inter-chain disulfide-linked peptide, an unambiguous marker of intact ricin. Dramatic enhancement of sensitivity in complex matrices was achieved through optimized immunocapture with sheep anti-mouse magnetic beads, enabling ∼80% recovery of ricin and compatibility with nano-LC-MS/MS acquisition at concentrations below 1 ng/mL. The proteomic method matched the sensitivity of the parallel adenine-release assay, with limits of detection (LOD) down to 10 pg/mL for the A-, B-chain peptides and adenine. The most abundant linker peptide variants were detected at 500 pg/mL. Good repeatability was achieved, with average CV values below 20% across analyses and matrices. This approach enables simultaneous assessment of chain connectivity and biological activity, increasing confidence in identification relative to conventional approaches and providing unique insights into the toxin's functionality.