Dataset Information

Identification of Unknown Biological Toxin Protein Using Mass Spectrometry: A Case Study on De Novo Sequencing of Ricin

ABSTRACT: Currently, the genomes of many toxin proteins remain unsequenced. Characterizing these toxin proteins and their active peptide segments is crucial for the development of detoxification agents and for discovering proteins with potential therapeutic applications. Existing methods for identifying and detecting toxin proteins primarily focus on known proteins, making them less applicable for identifying toxins from unknown sources. In this study, we employed a combination of multiple enzymatic digestion techniques, de novo mass spectrometry sequencing, and sequence assembly methods based on graph theory to perform high-precision, full-length sequencing of multi-subunit toxin proteins. After de novo sequencing and identifying Ricin toxin proteins, we utilized a strategy based on multiple alignments and error correction through the construction of overlapping scaffolds to differentiate between homologous isomers I and L and to detect unexpected post-translational modifications. This approach, referred to as the Heuristic Denovo protein sequencing(HDPS), facilitated the rapid hydrolysis and identification of proteins. The method achieved a 100% coverage rate for protein/peptide biological toxins and an amino acid-level accuracy of over 95%. Through de novo sequencing of Ricin toxin, this study provides a stable and reliable solution for identifying highly mutated proteins and those from unknown sequence sources.

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ORGANISM(S): Ricinus Communis

SUBMITTER: Yubo Song

LAB HEAD: Fuli Wang

PROVIDER: PXD061213 | Pride | 2025-12-01

REPOSITORIES: Pride

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Identification of Unknown Biological Toxin Proteins Using Mass Spectrometry: A Case Study on De Novo Sequencing of Ricin.

Song Yubo Y Wang Hao H Wen Junjie J Xu Jiale J Zhu Siyu S Wang Fuli F Zhang Yongqian Y

Toxins 20251118 11

<h4>Background</h4>The rapid and reliable identification of unknown or highly variable biological toxin proteins, such as the potent Ricin toxin, remains a critical challenge in biodefense and public security.<h4>Methods</h4>To address this, we developed a Heuristic De Novo Sequencing (HDPS) strategy, which combines multiple enzymatic and microwave-assisted acid hydrolysis to generate diverse peptides, followed by a two-stage assembly process integrating de novo sequencing with homology-based da ...[more]

PMID: 41295879

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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.

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	1_ProteinaseK1.raw	Raw
	2_ProteinaseK2.raw	Raw
	3_Glu-C.raw	Raw
	4_Chymotrysin.raw	Raw
	5_Trypsin.raw	Raw