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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 Chromatog-raphy-Tandem Mass Spectrometry

ABSTRACT: 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 speci-ficity. 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 rela-tive to a standard, namely 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 the MS signals for 15N and natural isotopic abundance ricin peptides are distinct, with mass shifts that correspond to the numbers of nitrogen atoms in each peptide or fragment. We also show that, as expected, labeled and unlabeled peptides coelute, with relative retention time differences of less than 0.2%.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Ricinus Communis

TISSUE(S): Seed

SUBMITTER: Eric Merkley

LAB HEAD: Eric Merkley

PROVIDER: PXD015698 | Pride | 2020-07-16

REPOSITORIES: Pride

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			Action	DRS
	Rcom_15N_13A_7Dec18_Merry_18-09-02.mzML	Mzml
	Rcom_15N_13A_7Dec18_Merry_18-09-02.mzid.gz	Mzid
	Rcom_15N_13A_7Dec18_Merry_18-09-02.pride.mgf.gz	Mgf
	Rcom_15N_13A_7Dec18_Merry_18-09-02.pride.mztab.gz	Mztab
	Rcom_15N_13A_7Dec18_Merry_18-09-02.raw	Raw

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Project description:The toxin ricin has been shown to cause inflammatory lung damage, leading to pulmonary oedema and, at higher doses, mortality. In order to understand the genetic basis of this inflammatory cascade a custom microarray platform (1509 genes) directed towards immune and inflammatory markers was used to investigate the temporal expression profiles of genes in a Balb/c mouse model of inhalational ricin exposure. To facilitate examination of those genes involved in both inflammatory cascades and wound repair the dose which was investigated was sub-lethal across a 96 hour time course. Histopathology of the lung was mapped across the time course and genetic responses considered in the context of overall lung pathology. 685 genes were found to be statistically significantly different compared to controls, across the time course and these genes have been investigated in the context of their biological function in ricin poisoning. As well as confirming key inflammatory markers associated with ricin intoxication (TNFa and Il1b) several pathways that are altered in expression were identified following pulmonary exposure to ricin. These genes included those involved in cytokine-cytokine signalling cascades (IL1, IL1r, IL1r2, Ccl 4, 6, 10), focal adhesion (Fn1, ICAM1) and tissue remodelling (VEGF, Pim1). Furthermore, the observed alteration in expression of matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) indicates a key role in membrane integrity and cellular adhesion in ricin poisoning. Data captured using this transcriptomic approach could be used to develop a specific approach to the treatment of inhalational ricin exposure. This work was conducted as part of a wider programme of work to compare a number of militarily relevant lung damaging agents, with a view to establishing a rational basis for the identification of more generic medical countermeasures. Four groups of six mice were exposed to ricin and a further groups exposed to vehicle only control (PBS). Following exposure mice were culled at 6 timepoints (1, 4, 7, 24, 48 and 96h). RNA was extracted and run on the custom array.

Dataset Information

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 Chromatog-raphy-Tandem Mass Spectrometry

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