SCX-tip-aided LC-MS detection of active ricin<i>via</i>oligonucleotide substrates for depurination kinetics

Yang, Zhaohui; Wang, Chenyu; Xiao, Lan; Wang, Chuang; Li, Tang; Xie, Jian‐Wei

doi:10.1039/d3an00217a

Cited by 3 publications

(2 citation statements)

References 47 publications

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“…Based on our previous work on the molecular docking between ricin and oligonucleotides substrates [ 27 , 28 ], in which 13 residues composing primary and secondary pockets of ricin were chosen, and the fact that the docking results meet the experimental well, as a result of this, the same docking parameters applied to the interaction between peptide and ricin. In the H-dock, the fix of the primary and secondary pockets of ricin is reasonable because we also performed a total “blind” docking of the peptide to ricin in Discovery Studio without setting receptor binding site residues.…”

Section: Resultsmentioning

confidence: 99%

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In Silico–Ex Vitro Iteration Strategy for Affinity Maturation of Anti-Ricin Peptides and the SPR Biosensing Application

Yang

Wang

Liu

et al. 2023

Toxins

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The highly toxic plant toxin ricin is one of the most known threatening toxins. Accurate and sensitive biosensing methods for the first emergency response and intoxication treatment, are always pursued in the biodefense field. Screening affinity molecules is the fundamental mainstream approach for developing biosensing methods. Compared with common affinity molecules such as antibodies and oligonucleotide aptamers, peptides have great potential as biosensing modules with more accessible chemical synthesis capability and better batch-to-batch stability than antibodies, more abundant interaction sites, and robust sensing performance towards complex environments. However, anti-ricin peptides are so scant to be screened and discovered, and an advanced screening strategy is the utmost to tackle this issue. Here, we present a new in silico-in vitro iteration-assisted affinity maturation strategy of anti-ricin peptides. We first obtained affinity peptides targeting ricin through phage display with five panning rounds of “coating-elution-amplification-enrichment” procedures. The binding affinity and kinetic parameters characterized by surface plasmon resonance (SPR) showed that we had obtained four peptides owning dissociation constants (KD) around 2~35 μM, in which peptide PD-2-R5 has the lower KD of 4.7 μM and higher stable posture to interact with ricin. We then constructed a new strategy for affinity maturity, composing two rounds of in silico-in vitro iterations. Firstly, towards the single-site alanine scanning mutation peptide library, the molecular docking predictions match the SPR evaluation results well, laying a solid foundation for designing a full saturation mutated peptide library. Secondly, plenty of in silico saturation mutation prediction results guided the discovery of peptides PD2-R5-T3 and PD-2-R5-T4 with higher affinity from only a limited number of SPR evaluation experiments. Both evolved peptides had increased affinity by about 5~20 times, i.e., KD of 230 nM and 900 nM. A primary cellular toxicity assay indicated that both peptides could protect cells against ricin damage. We further established an SPR assay based on PD-2-R5-T3 and PD-2-R5-T4 elongated with an antifouling peptide linkage and achieved good linearity with a sensitivity of 1 nM and 0.5 nM, respectively. We hope this new affinity-mature strategy will find its favorable position in relevant peptide evolution, biosensing, and medical countermeasures for biotoxins to protect society’s security and human life better.

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Section: Resultsmentioning

confidence: 99%

“…Ricin, RCA120, abrin, and AAG with milligram amounts were prepared in-house with an electrophoretic purity greater than 95% [ 27 ]. Peptides and biotinylated peptides were synthesized and characterized by Synpeptide Co., Ltd. (Nanjing, China).…”

Section: Methodsmentioning

confidence: 99%

In Silico–Ex Vitro Iteration Strategy for Affinity Maturation of Anti-Ricin Peptides and the SPR Biosensing Application

Yang

Wang

Liu

et al. 2023

Toxins

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Simultaneous analysis of depurinated nucleic acid stem-loop and free adenine for ricin toxicity assay by hydrophilic interaction liquid chromatography-high-resolution mass spectrometry (HILIC-HRMS)

Miyaguchi

2024

Anal. Methods

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A Glycoprotein-Based Surface-Enhanced Raman Spectroscopy–Lateral Flow Assay Method for Abrin and Ricin Detection

Xiao,

Luo,

Liu

et al. 2024

Toxins

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Abrin and ricin, both type II ribosome-inactivating proteins, are toxins of significant concern and are under international restriction by the Chemical Weapons Convention and the Biological and Toxin Weapons Convention. The development of a rapid and sensitive detection method for these toxins is of the utmost importance for the first emergency response. Emerging rapid detection techniques, such as surface-enhanced Raman spectroscopy (SERS) and lateral flow assay (LFA), have garnered attention due to their high sensitivity, good selectivity, ease of operation, low cost, and disposability. In this work, we generated stable and high-affinity nanotags, via an efficient freezing method, to serve as the capture module for SERS-LFA. We then constructed a sandwich-style lateral flow test strip using a pair of glycoproteins, asialofetuin and concanavalin A, as the core affinity recognition molecules, capable of trace measurement for both abrin and ricin. The limit of detection for abrin and ricin was 0.1 and 0.3 ng/mL, respectively. This method was applied to analyze eight spiked white powder samples, one juice sample, and three actual botanic samples, aligning well with cytotoxicity assay outcomes. It demonstrated good inter-batch and intra-batch reproducibility among the test strips, and the detection could be completed within 15 min, indicating the suitability of this SERS-LFA method for the on-site rapid detection of abrin and ricin toxins.

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SCX-tip-aided LC-MS detection of active ricinviaoligonucleotide substrates for depurination kinetics

Abstract: The accurate, sensitive detection of active biotoxin proteins and thereafter their kinetics feature is vital for the upsurge of chemical attacks but is still limited. Here we report a liquid...

Cited by 3 publications

References 47 publications

In Silico–Ex Vitro Iteration Strategy for Affinity Maturation of Anti-Ricin Peptides and the SPR Biosensing Application

In Silico–Ex Vitro Iteration Strategy for Affinity Maturation of Anti-Ricin Peptides and the SPR Biosensing Application

Simultaneous analysis of depurinated nucleic acid stem-loop and free adenine for ricin toxicity assay by hydrophilic interaction liquid chromatography-high-resolution mass spectrometry (HILIC-HRMS)

A Glycoprotein-Based Surface-Enhanced Raman Spectroscopy–Lateral Flow Assay Method for Abrin and Ricin Detection

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