Rapid analysis of ricin using hot acid digestion and MALDI‐TOF mass spectrometry

Chen, Dapeng; Bryden, Wayne A.; Fenselau, Catherine

doi:10.1002/jms.4257

Cited by 14 publications

(10 citation statements)

References 21 publications

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“…For the MS-based detection of protein toxins, trypsin digestion is the most time-consuming step, which normally takes up to 18–24 h [ 19 ]. To address these, some strategies were employed, such as immobilized trypsin [ 20 ], organic solvent assistance, surfactant-assisted protocols [ 21 , 22 ], hot acid digestion [ 23 ], microwave- [ 24 ], and ultrasound-assisted protocols [ 18 ]. As we know, protein denaturation and unfolding is a crucial step during digestion, especially for ricin and abrin that are resistant to proteases due to their compact structure.…”

Section: Resultsmentioning

confidence: 99%

Rapid Differential Detection of Abrin Isoforms by an Acetonitrile- and Ultrasound-Assisted On-Bead Trypsin Digestion Coupled with LC-MS/MS Analysis

Liang¹,

Yang²,

Geng³

et al. 2021

Toxins

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The high toxic abrin from the plant Abrus precatorius is a type II ribosome-inactivating protein toxin with a human lethal dose of 0.1–1.0 µg/kg body weight. Due to its high toxicity and the potential misuse as a biothreat agent, it is of great importance to developing fast and reliable methods for the identification and quantification of abrin in complex matrices. Here, we report rapid and efficient acetonitrile (ACN)- and ultrasound-assisted on-bead trypsin digestion method combined with HPLC-MS/MS for the quantification of abrin isoforms in complex matrices. Specific peptides of abrin isoforms were generated by direct ACN-assisted trypsin digestion and analyzed by HPLC-HRMS. Combined with in silico digestion and BLASTp database search, fifteen marker peptides were selected for differential detection of abrin isoforms. The abrin in milk and plasma was enriched by immunomagnetic beads prepared by biotinylated anti-abrin polyclonal antibodies conjugated to streptavidin magnetic beads. The ultrasound-assisted on-bead trypsin digestion method was carried out under the condition of 10% ACN as denaturant solvent, the entire digestion time was further shortened from 90 min to 30 min. The four peptides of T3Aa,b,c,d, T12Aa, T15Ab, and T9Ac,d were chosen as quantification for total abrin, abrin-a, abrin-b, and abrin-c/d, respectively. The absolute quantification of abrin and its isoforms was accomplished by isotope dilution with labeled AQUA peptides and analyzed by HPLC-MS/MS (MRM). The developed method was fully validated in milk and plasma matrices with quantification limits in the range of 1.0-9.4 ng/mL for the isoforms of abrin. Furthermore, the developed approach was applied for the characterization of abrin isoforms from various fractions from gel filtration separation of the seeds, and measurement of abrin in the samples of biotoxin exercises organized by the Organization for the Prohibition of Chemical Weapons (OPCW). This study provided a recommended method for the differential identification of abrin isoforms, which are easily applied in international laboratories to improve the capabilities for the analysis of biotoxin samples.

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Section: Resultsmentioning

confidence: 99%

Rapid Differential Detection of Abrin Isoforms by an Acetonitrile- and Ultrasound-Assisted On-Bead Trypsin Digestion Coupled with LC-MS/MS Analysis

Liang¹,

Yang²,

Geng³

et al. 2021

Toxins

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“…Bacillus spores were suspended in 100 μL of 12.5% acetic acid solution and the digestion was conducted at 140 °C for 15 min. The selection of pH, temperature and time for these experiments was based on previous optimization studies in this laboratory [9,13,14]. After hot acid hydrolysis, samples were centrifuged at 10,000 × g for 1 min.…”

Section: Microwave-assisted Hot Acid Digestionmentioning

confidence: 99%

“…However, the similarity in SASP sequences and masses suggests that additional or alternative proteins should also be investigated as biomarkers [8]. In previous studies, we established a microwave-assisted hot acid method that provides extensive protein hydrolysis [9,10]. In this study, the method is applied to lyse four Bacillus spore types: B. globigii, B. thuringiensis (Hakam), B. anthracis (sterne), and B. subtilis (168) and to proteolyse a wider selection of released proteins.…”

Section: Introductionmentioning

confidence: 99%

Microwave supported hydrolysis prepares Bacillus spores for proteomic analysis

Chen

Bryden

Fenselau

2019

International Journal of Mass Spectrometry

Self Cite

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Rapid identification of Bacillus spores in the environment has depended primarily on a family of small acid soluble proteins (SASPs) as biomarkers. However, SASP sequences and molecular masses are similar or identical in some critical cases. For example, some strains of B. subtilis, and B. thuringiensis cannot be distinguished from strains of B. anthracis based on SASPs. Consequently, additional or alternative biomarkers should be sought. In this study microwaveassisted hot acid hydrolysis was coupled with mass spectrometry as a potentially powerful approach to the rapid automatable characterization of Bacillus spores. Hot acid provides lysis of the spores, Asp-selective hydrolysis of proteins, and peptides compatible with automated analysis of either peptide fingerprints or tandem mass spectra. Peptide biomarkers are compared here for a selection of Bacillus spores, and peptides unique to each spore type are identified.

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“…17 Residuespecific protein cleavage using microwave-assisted hot acid hydrolysis provide a novel strategy to characterize ricin by matrix-assisted laser desorption ionization-time of flight analysis. 18 Direct tryptic digestion of intact ricin into peptides had been tried to reduce the total time, but ricin in the aqueous buffer is highly resistant to trypsin digestion. 13 Long time and large amounts of enzyme usage are required for digestion to get enough tryptic peptides in our previous study.…”

Section: Introductionmentioning

confidence: 99%

Direct Acetonitrile-Assisted Trypsin Digestion Method Combined with LC–MS/MS-Targeted Peptide Analysis for Unambiguous Identification of Intact Ricin

Liu¹,

Liang²,

Yang³

et al. 2020

J. Proteome Res.

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Ricin is a type II ribosome-inactivating protein toxin consisting of A and B chains linked by one interchain disulfide bond. Because of its high toxicity depending on both chains together, confirming the presence of both A and B chains of intact ricin is required during the investigation of the illegal production and application. Here, we report a novel and sensitive acetonitrile (ACN)-assisted trypsin digestion method for unambiguous identification of intact ricin by simultaneous detection of its marker peptides from A and B chains. Marker peptides were generated with a simple procedure by direct cleaving the native ricin at 45 °C for 4 h using Promega modified sequencing grade trypsin under the assistance of 10% ACN, and then directly analyzed by ultrahigh performance liquid chromatography tandem mass spectrometry. The type of trypsin was found to be one critical factor for cleavage of intact ricin based on a significant difference in the yields of specific peptides generated while using various types of trypsin. A low content of ACN in enzymatic buffer significantly reduced the digestion time from overnight to 4 h. There was commonly a better MS response of marker peptides when using the developed ACN-assisted trypsin digestion method than methanol-assisted trypsin digestion within the same 4 h. Totally, seven specific peptides with high sensitivity and specificity including three in the A-chain (TA7, TA11, and TA10) and four in the B-chain (TB6, TB14-ss-TB16, TB20, and TB18) were obtained as good marker peptides for unambiguous identification of intact ricin. The lowest concentration of native ricin for unambiguous identification was 20 ng/mL, in which three marker peptides from both the A-chain and B-chain could be measured with a minimum of three ion transitions. Combined with affinity enrichment, the developed approach was successfully applied for the measurement of intact ricin from the complicated matrix samples of the second, third, and fourth biotoxin exercises organized by the Organisation for the Prohibition of Chemical Weapons (OPCW). This study has provided a recommended detection method combined with one novel ACN-assisted trypsin digestion with MS for forensic unambiguous confirmation of trace ricin intact with high confidence.

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Rapid analysis of ricin using hot acid digestion and MALDI‐TOF mass spectrometry

Cited by 14 publications

References 21 publications

Rapid Differential Detection of Abrin Isoforms by an Acetonitrile- and Ultrasound-Assisted On-Bead Trypsin Digestion Coupled with LC-MS/MS Analysis

Rapid Differential Detection of Abrin Isoforms by an Acetonitrile- and Ultrasound-Assisted On-Bead Trypsin Digestion Coupled with LC-MS/MS Analysis

Microwave supported hydrolysis prepares Bacillus spores for proteomic analysis

Direct Acetonitrile-Assisted Trypsin Digestion Method Combined with LC–MS/MS-Targeted Peptide Analysis for Unambiguous Identification of Intact Ricin

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