Rapid Differential Detection of Abrin Isoforms by an Acetonitrile- and Ultrasound-Assisted On-Bead Trypsin Digestion Coupled with LC-MS/MS Analysis
| Autor: | Chang-Cai Liu, Yang Yang, Hui-Lan Yu, Long-Hui Liang, Shu Geng, Shi-Lei Liu, Xi Cheng |
|---|---|
| Rok vydání: | 2021 |
| Předmět: |
Streptavidin
RIP II protein toxins Health Toxicology and Mutagenesis Size-exclusion chromatography Isotope dilution Toxicology medicine.disease_cause 01 natural sciences Article 03 medical and health sciences chemistry.chemical_compound Tandem Mass Spectrometry Abrus precatorius HPLC-ESI-MS/MS medicine Animals Protein Isoforms Computer Simulation Trypsin Ultrasonics Chromatography High Pressure Liquid 030304 developmental biology Toxins Biological 0303 health sciences Chromatography biology Toxin 010401 analytical chemistry biology.organism_classification 0104 chemical sciences Milk chemistry Polyclonal antibodies ultrasound-assisted trypsin digestion Biotinylation Abrus biology.protein Medicine marker peptides Abrin Rabbits detection of abrin Chromatography Liquid |
| Zdroj: | Toxins Toxins, Vol 13, Iss 358, p 358 (2021) Volume 13 Issue 5 |
| ISSN: | 2072-6651 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|