Uniformly 15N-Labeled Recombinant Ricin A-chain as an Internal Retention Time Standard for Increased Confidence in Forensic Identification of Ricin by Untargeted Nanoflow Liquid Chromatography-Tandem Mass Spectrometry

Ricin, a toxic protein from the castor plant, is of forensic and biosecurity interest because of its high toxicity and common occurrence in crimes and attempted crimes. Qualitative methods to detect ricin are therefore needed. Untargeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) proteomics methods are well suited because of their high specificity. Specificity in LC-MS/MS comes from both the LC and MS components. However, modern untargeted proteomics methods often use nanoflow LC, which has less reproducible retention times than standard-flow LC, making it challenging to use retention time as a point of identification in a forensic assay. We address this challenge by using retention times relative to a standard, namely, the uniformly 15N-labeled ricin A-chain produced recombinantly in a bacterial expression system. This material, added as an internal standard prior to trypsin digestion, produces a stable-isotope-labeled standard for every ricin tryptic peptide in the sample. We show that th...