Ricin, a ribosomal inactivating protein toxin produced by the castor plant (Ricinus communis) is considered a biological threat due to its accessibility and low human lethal dose. Method-based mass spectrometry (MS) techniques for the identification of protein toxins such as Ricin are needed to enhance analytical processes for data collection on bioforensic samples. MS-based identification of Ricin involves comparison of MS measurements between test samples and reference standards in addition to identification of Ricin-specific peptides by searching proteome databases. The National Bioforensic Analysis Center (NBFAC) validated two MS techniques used to identify Ricin in bioforensic samples following the ISO 17025 Standard. These methods include: 1) 1D SDS-PAGE, for separation and identification by molecular weight, trypsin digestion and MALDI TOF/TOF analysis and 2) in solution trypsin digestion and nanoLC-HRMS/MS analysis. Robust method acceptance criteria were established for ISO 17025 validation to provide confidence in results. Protein identification is made after all MS data are searched through commercially-available proteomics software, compared to data obtained from reference standards analyzed in parallel, and manually reviewed to confirm peptide identifications. The final protein identification is made when at least one peptide unique to the protein toxin is identified. This ISO 17025 validation was performed over a year by multiple technicians providing established limits of detections, sensitivity, false positive/negative rates and variability for the methods. Processing of non-toxin ribosomal inactivating proteins demonstrated that the methods are specific to the identification of Ricin. ISO 17025 accreditation of these MS techniques for Ricin identification provides confidence in the robust results.

ISO 17025 validation of method-based mass spectrometry techniques for the identification of Ricin in bioforensic samples

256th National Meeting (2018)

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