Rapid Detection of Abrin Toxin and Its Isoforms in Complex Matrices

Oct 6, 2017 - Biological Toxins, Centre for Biological Threats and Special Pathogens, Robert Koch Institute, Berlin, Germany. § Service de Pharmacolo...
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Rapid detection of abrin toxin and its isoforms in complex matrices by immuno-extraction and quantitative high resolution targeted mass spectrometry Eva-Maria Hansbauer, Sylvia Worbs, Hervé Volland, Stéphanie Simon, Christophe Junot, François Fenaille, Brigitte G. Dorner, and Francois Becher Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b03189 • Publication Date (Web): 06 Oct 2017 Downloaded from http://pubs.acs.org on October 10, 2017

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Analytical Chemistry

Rapid detection of abrin toxin and its isoforms in complex matrices by immuno-extraction and quantitative high resolution targeted mass spectrometry Eva-Maria Hansbauer,† Sylvia Worbs,║ Hervé Volland,‡ Stéphanie Simon‡, Christophe Junot, ₸ François Fenaille,† Brigitte G. Dorner, ║ François Becher†* † Service de Pharmacologie et Immunoanalyse (SPI), Laboratoire d'Etude du Métabolisme des Médicaments, CEA, INRA, Université Paris Saclay, F-91191 Gif-sur-Yvette cedex, France ║ Biological Toxins, Centre for Biological Threats and Special Pathogens, Robert Koch Institute, Berlin, Germany ‡ Service de Pharmacologie et Immunoanalyse (SPI), Laboratoire d'Etudes et de Recherches en Immunoanalyse, CEA, INRA, Université Paris Saclay, F-91191 Gif-sur-Yvette cedex, France ₸ Service de Pharmacologie et Immunoanalyse (SPI), CEA, INRA, Université Paris Saclay, F-91191 Gif-sur-Yvette cedex, France.

ABSTRACT: Abrin expressed by the tropical plant Abrus precatorius is highly dangerous with an estimated human lethal dose of 0.1–1 µg/kg body weight. Due to the potential misuse as a biothreat agent, abrin is in the focus of surveillance. Fast and reliable methods are therefore of great importance for early identification. Here, we have developed an innovative and rapid multi-epitope immuno-mass spectrometry workflow which is capable of unambiguously differentiating abrin and its isoforms in complex matrices. Toxin containing samples were incubated with magnetic beads coated with multiple abrin-specific antibodies, thereby concentrating and extracting all the isoforms. Employing an ultrasonic bath for digestion enhancement, on-bead trypsin digestion was optimized to obtain efficient and reproducible peptide recovery in only 30 min. Improvements made to the workflow reduced total analysis time to less than 3 hours. A large panel of common and isoform-specific peptides was monitored by multiplex LC-MS/MS through the parallel reaction monitoring mode on a quadrupole-Orbitrap high resolution mass spectrometer. Additionally, absolute quantification was accomplished by isotope dilution with labeled AQUA peptides. The newly established method was demonstrated as being sensitive and reproducible with quantification limits in the low ng/mL range in various food and clinical matrices for the isoforms of abrin and also the closely related, less toxic Abrus precatorius agglutinin. This method allows for the first time the rapid detection, differentiation and simultaneous quantification of abrin and its isoforms by mass spectrometry.

Highly poisonous biological toxins are in the focus of surveillance by international and national authorities due to the threat of their deliberate release. Among these is the plant toxin abrin, mentioned on the U.S. Select Agents and Toxins list, based on the ease of preparation and the high morbidity.1 Abrin is contained in the seeds of Abrus precatorius found in tropical regions around the world.2 It belongs, as well as the plant toxin ricin from Ricinus communis, to the ribosomal inactivating protein class II (RIP II) and has attracted much attention regarding criminal and terroristic misuse over the last decade.3 Both ricin and abrin are highly dangerous to humans, with similar minimum lethal doses at 2.7 and 0.7 µg/kg body weight after i.v. injection, respectively.4 Symptoms of intoxication are non-specific and may include vomiting, diarrhoea, nausea, fever, sore throat or respiratory disorder depending on the route of intoxication.3,5,6 The molecular basis of these symptoms is cellular apoptosis induced by an arrest of protein synthesis due to deadenylation of ribosomal RNA in the so-called α-sarcin/ricin loop of 28S subunit of ribosomes after galactose-mediated endocytosis of RIP II toxins.7–11 Abrin is a 60 kDa protein consisting of an enzymatic active Achain and a receptor-binding B-Chain, connected by a disulphide bond, and is a complex protein in terms of structural modifications.7,12 In addition to two N-glycosylation sites on the B-

chain, four different isoforms of abrin have been isolated from the seeds of Abrus precatorius.12,13 Abrin-a, -b, -c and -d resemble each other with 78% protein identity; in contrast to the two ricin D and E isoforms, amino-acid variations in abrin are distributed along the sequence.14 Different biochemical properties of abrin isoforms, like the ability to bind sepharose, were experimentally detected, resulting in differences in lethal doses ranging from 10 to 31 µg/kg body weight (i.p. injected).12,15 Abrin shares high sequence homology with Abrus agglutinin (67% sequence identity in the A-Chain and 80% in the B-Chain on amino acid level) from the same seeds. Although Abrus agglutinin contains the same RNA N-Glycosidase activity, it is several magnitudes less toxic than abrin due to structural changes in the active site.16,17 Up to now, the relative proportions of abrin isoforms and agglutinin in the seeds remain unclear, as well as alterations in protein composition in material obtained from different geographical origins.18 Reliable and fast identification of toxins in potentially contaminated environmental or clinical samples at low level is of great importance, especially in a potential bioterror threat scenario. Only few techniques were reported for the detection of abrin, mainly based on three different markers contained in the seeds of A. precatorius: DNA, the surrogate alkaloid L-Abrine or the protein itself.19–21 The latter is the preferable way of detection

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since both DNA and L-Abrine will probably be undetectable in cases of intoxication with purified toxin. Current abrin protein detection is mainly based on antibody-based technologies. Antibodies can be employed for stationary methods like ELISA or fluorescent multiplex bead arrays.22–24 Furthermore, on-site detection methods, e.g. lateral flow assays, are suitable for fast and preliminary risk assessment.25,26 Generally, antibody-based methods are among the most sensitive methods available provided that high-affinity tools are used for detection. It has to be considered that antibodies can generate either false-positive or false-negative results due to e.g. unspecific binding to matrix components or variable binding to sequence variants of the target protein, respectively. 27,28 Therefore, results obtained by antibody-based technologies should be confirmed by methods revealing sequence information, such as mass spectrometry (MS).29 In the last few years, targeted MS-analyses of proteotypic peptides led to robust, sensitive and specific quantification of bioterrorist-related toxins.30–36 A drawback of these bottomup approaches is the time-consuming step of protein digestion into peptides. Protocols to shorten total assay time while preserving high levels of sensitivity and quantitative accuracy are essential in case of a bioterrorist threat for rapid discrimination of the agent. Still, the establishment of targeted assays to complex proteins remains challenging and relies on the proper selection of protein enrichment conditions and of surrogate proteotypic peptides.32,37–39 In this report, we describe a rapid mass spectrometry-based method monitoring a large panel of common and isoform-specific abrin peptides in complex matrices to ensure sensitive and unambiguous identification of abrin and its isoforms. A multiplex enrichment step was implemented to extract all the targeted isoforms as well as the surrogate marker agglutinin prior to mass spectrometry for improved detection in a wide range of complex environmental or biological matrices that may be investigated in a biodefense scenario. Additionally, improvements to the workflow, especially through efficient ultrasonic assisted trypsin digestion, reduced total analysis time from two days to three hours. As a final step, multiplex, sensitive, and reproducible quantification was achieved through the parallel reaction monitoring (PRM) mode available on a quadrupoleOrbitrap high resolution instrument. This method allows for the first time the rapid and simultaneous identification and quantification of abrin and its different isoforms by targeted mass spectrometry.

EXPERIMENTAL SECTION Safety precaution. Due to the high toxicity, experiments using abrin were performed in a biosafety level-2 cabinet equipped with a HEPA filter. Only trained personnel was allowed to handle the toxin while wearing personal protection equipment and following specified safety protocols. RIP-contaminated solutions and consumables were inactivated overnight using 2 M NaOH. Chemicals and material. Abrus seeds were purchased from Sandeman Seeds (West Sussex, United Kingdom). Sequencing grade modified trypsin was obtained from Promega Corporation (Charbonnières-les-Bains, France). RapiGest SF Surfactant was purchased from Waters Corporation (Milford, USA). Dynabeads M-280 tosylactivated magnetic beads were obtained from Invitrogen (Life Technologies, Oslo, Norway). Labeled peptides for quantification were synthesized in Absolute

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QUAntitation (AQUA) ultimate quality by ThermoFisher Scientific (Paisley, UK). Water (ChromaSolve LC-MS), acetonitrile (HPLC-grade), and formic acid were obtained from Honeywell/Riedel-de Haen (Seelze, Germany) and VWR chemicals (Fontenay sous Bois, France), respectively. All other chemicals were purchased from Sigma-Aldrich (Saint Quentin Fallavier, France) or VWR chemicals (Fontenay sous Bois, France). For all reactions, LoBind Eppendorf tubes (Dutscher, Brumath, France) were used.

Abrin extract. Abrin was purified from Abrus precatorius seeds as described earlier.12,40,41 Briefly, proteins were extracted overnight with acetic acid and precipitated with ammonium sulphate. Affinity chromatography with a Lactosyl-sepharose column was employed to isolate abrin isoforms and agglutinin. Purity (>95%) was checked by SDS-PAGE. The concentration in abrin/agglutinin of the lactosyl-purified material was determined by absorption at 280 nm with NanoPhotometer P300 (IMPLEN, Munich, Germany, ε = 1.525 mL×mg-1×cm-1 for abrin/agglutinin mixture).42 Protein sequence information. Sequence information was obtained from the uniprot database (http://www.uniprot.org) for abrin-a, -b, -c, and -d, and Abrus precatorius agglutinin:  entries P11140, Q06077, P28590, Q06076, and Q9M6E9, respectively (Figure S1, Supporting Information). Characterization of purified abrin. For characterization of abrin isoforms, 1.46 µg lactosyl-purified abrin was diluted with 19 µL RapiGest SF (0.05% in 50 mmol/L ammonium bicarbonate) and 10 µL of 3 mg/mL dithiothreitol. It was heated for 15 min at 95°C to induce denaturation. After cooling down to room temperature, 10 µL of 4.5 mg/mL iodacetamide was added for alkylation and incubated 45 min in the dark. Then, 2.5 µg of endoproteinase GluC (5 µL of 0.5 µg/µL in 50 mM Ammonium bicarbonate), 0.1 µg of endoproteinase AspN (5 µL of 0.02 µg/µL in 50 mM Ammonium bicarbonate) and/or 1 µg sequencing grade modified Trypsin (5 µL of 0.2 µg/µL in H2O) were used for digestion at 37°C in the following combinations: only one protease or a sequential digestion with AspN or GluC overnight followed by an overnight digest with Trypsin. The digests were stopped by adding 5 µL of 1M HCl. Moreover, HCl degraded the surfactant RapiGest SF after 45 min incubation at 37°C. For quantification of abrin isoforms, 1.46 µg purified abrin was diluted with 66 µL 0.05% RapiGest SF and heated for 15 min at 95°C. After cooling down to room temperature, 1.5 µg of sequencing grade modified trypsin were added. The samples were incubated in a standard heating block at 37°C for 6 hours. The tryptic digest was stopped by adding 30 µL of 1M HCl followed by 45 min of incubation for surfactant degradation. The solution was diluted 1/2.5 with MS buffer (95% H2O, 5% acetonitrile, 0.1% formic acid). Increasing concentrations (1 fmol/µL–0.2 pmol/µL) of AQUA peptides in MS buffer were added to generate a standard curve in subsequent liquid chromatography and mass spectrometry (LC-MS/MS) analyses. The extracted ion chromatogram (XIC) peak area ratio of tryptic native peptides to AQUA peptides was used for the back-calculation of abrin isoforms concentrations. Immunocapture of abrin in matrices. Antibodies used for abrin capture were produced in-house at RKI using a formaldehyde inactivated mixture of abrin and agglutinin for immunization of mice.24 On-beads immobilization was done according to

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the manufacturer’s instruction using 140 µg antibody coupled to 14.8 mg tosylactivated Dynabeads. Five hundred microliters of toxin-containing samples were incubated with 0.211 µg antibody-coated magnetic beads (corresponding to 2 µg antibody per reaction) for 1 hour under rotation. The tubes were placed in a magnet and the supernatant was removed. The beads were sequentially washed with 0.5 mL PBS/Tween 0.05%, 0.25 mL PBS and 0.25 mL H2O. The dry beads were resuspended in 10 µL RapiGest SF (0.05% in 50 mmol/L ammonium bicarbonate) and 5 µl H2O.

On-beads tryptic digest. The bead samples were heated for 15 min at 95°C for denaturation. After cooling down to room temperature, 0.5 µg of sequencing grade modified trypsin (2.5 µL of 0.2 µg/µL in H2O) was added. The samples were placed in a bath-type sonicator (Advantage Lab, Darmstadt, Germany) at 35°C for 30 min. Overnight incubation and 2 h incubation at 37°C in a conventional thermocycler were evaluated in parallel during optimization of tryptic digest. The digest was stopped by adding 5 µL of 1 M HCl followed by 45 min of incubation at 37°C for surfactant degradation. The samples were centrifuged (13000 rpm, 10 min); the supernatant was used for subsequent LC-MS/MS analyses following addition of 0.33 pmol of the 12 labeled AQUA peptides in MS buffer. Liquid chromatography and mass spectrometry. LCMS/MS data were acquired using a Dionex Ultimate 3000 system coupled to a Q-Exactive mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) using an Aeris peptide XB-C18 reverse phase column (150 x 2.1mm; 1.7 µm; 100 Å; phenomex, Le Pecq, France). The column oven temperature was set to 60°C. For peptide LC separation, 10 µL of the samples were injected. Mobile phases consisting of LC-MS grade water with 0.1% formic acid (phase A) and acetonitrile with 0.1% formic acid (phase B) were delivered at a flow rate of 0.5 mL/min. For abrin quantification by targeted MS, total run time of the method was 13 min. After an isocratic step of 1.5 min at 5% phase B, a linear gradient was applied ramping from 5% to 36.7% B between 1.5 and 9.2 min. The concentration of phase B increased to 95% during the next 0.3 min and was constant for 1.7 min before reverting to initial condition of 5% B. Eluted peptides were introduced to the Q-Exactive instrument, operating in positive ion mode under time-scheduled sequential PRM acquisition, by electrospray ionization (ESI). Instrument parameters of the atmospheric pressure source were as follows: sheath gas flow rate 70, spray voltage 4kV, capillary temperature 320°C. Precursor ions from native and internal standard peptides were selected in the quadrupole (confer Table S1, Supporting Information) with an isolation mass window of 1.5 m/z. They were fragmented in the HCD cell using nitrogen as collision gas and the given normalized collision energy (Table S1, Supporting Information). All fragment ions were transferred to the Orbitrap. Resolution was set to 35’000 at m/z 200 (full width at half maximum), automatic gain control to 1e6 and maximum injection time to 125 ms during 1.8–5.2 min and 6.8– 9.3 min. For 5.2–6.8 min, parameters were changed due to high number of peptides eluting in this timeframe: resolution was 17’000, automatic gain control was 1e6, and maximum injection time was set to 85 ms. Xcalibur 2.2 software (Thermo Fisher Scientific, Bremen, Germany) was used for instrument control and to process the data files.

For identification of proteotypic peptides by bottom-up experiment, the following parameters were applied: the run time was extended to 60 min with a linear gradient reaching 40% at 50 min, followed by 3 min at 95% B. A full MS/ddMS² (Top 10) scan was used for data acquiring with a resolution of 70’000 (Full MS) or 17’500 (ddMS²) at m/z 200 (full width at half maximum) and normalized collision energy fixed to 18%. The automatic gain control was 1e6, and maximum injection time was set to 250 ms.

Data analysis for identification and quantification. Peptides were identified by their corresponding MS/MS fragments (Figure S2, Supporting Information), between three and seven fragments were taken into account in the PRM method (additionally the M+1 fragment; confer Table S1, Supporting Information). Strict coelution of the labeled and native/unlabeled versions of each peptide was verified. Non-interfered signals from selected fragments were summed to provide one extracted ion chromatogram (XIC) of each targeted peptide.31 An 8-point standard curve was generated for each matrix between 5 ng/mL and 500 ng/mL, based on the measured concentration of the purified extract (corresponding to 2.0 ng/mL– 202.6 ng/mL total abrin or 3.1 ng/mL–307.5 ng/mL agglutinin). Concentrations of 8.1 ng/mL, 60.8 ng/mL and 121.6 ng/mL abrin were applied in five replicates for the quality control (QC) samples. For method evaluation, the XIC peak area of the native unlabeled peptides was divided by the XIC peak area of the heavy peptides. Linear regression with 1/x weighting was applied to generate a standard curve. The coefficient of variation of QC samples in % (CV%) was determined on two days and should be below 20% for a set of measurements in the same matrix. Accuracy in % should be between 80–120% of the true value. Lower limit of quantification (LLOQ) was defined according to guidelines on bioanalytical methods validation as the lowest concentration with 0.97 (Figure 4 and Figure S4, Supporting Information). The fourth common peptide LEENQLWTLK was utilized only for confirmation for the presence of abrin due to lower efficient tryptic release. At the lower limit of quantification (LLOQ, definition see Material and methods, at 8.1 ng/mL) all four common peptides are detected. The quality control samples at this concentration showed accuracy of 84.5–110.4% and CV% below 18% for the quantitative peptides YEPTVR, QFIEALR and EQQWALYTDGSIR (Table 1).43 In additional experiments, the coefficient of variation during one day or within days was analyzed. The mean CV%intra and CV%inter were 9.4% and 11.8% at low concentration and 9.6% and 9.1% at high concentration (5 replicates for each concentration per day, Table 1). The limit of detection for total abrin was estimated as 2 ng/mL, 3 out of 4 common peptides of abrin could be measured with a XIC peak area greater than 3000. Other methods for abrin detection like LFA or ELISA reported comparable limits of detection,22,23,25 but they do not offer sequence confirmation and differentiation of isoforms.

1

rin

ab

rin

-a -b -c -a -d 1 ab -b rin -c -a -d 2 ab -b rin -c -a -d 3 -b -c -d ab 4 rin -a ab 1 rin -a ab 2 ab rin rin -b ab b-c rin -d ab c-d rin 1 ag -cgl d 2 u ag tini gl n 1 ut in in 2

0

ab

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Peptides

Figure 3. Comparison of tryptic digestion protocols. Mean peak area ratio of unlabeled to labeled peptides (n=2) are illustrated.

It turned out that the digestion for 30 min in the bath-type sonicator and 2 h in the heating block gave comparable area ratios. The overnight digest had a lower efficiency (up to factor 2.5), probably due to unspecific cleavages occurring during the long incubation time. The developed method allowed reducing the digestion time from overnight to 30 min. Since the digest in the bath-type sonicator was much faster, reproducible and shortened the analysis time, it was chosen for further experiments. Setting up a quantitative PRM detection based on rapid on-bead digestion was successful and decreased the total analysis time from 2 working days to 3 hours in total (Figure 2).

Evaluation of abrin quantification in milk. The newly established method was evaluated considering linearity, reproducibility and abrin stability. To this end, standard curves and quality control samples were tested. Based on the assumption, that potential abrin samples of biological concern would never occur in buffer, it was decided to record the evaluation in milk. Milk possesses a high protein and sugar content, especially the content of lactose could cause problems for the detection of lectin-binding toxins.52 With an 8-point standard curve prepared in milk, ranging from 2 ng/mL to 200 ng/mL for total abrin and 3 ng/mL to 300 ng/mL for agglutinin peptides, linearity of abrin and agglutinin quantification was evaluated (Figure 4 and Figure S4, Supporting Information). Each sample was immunopurified, digested in the ultrasonic bath, spiked with AQUA peptides and analyzed by the newly established PRM method (Figure 2). In addition, five replicates of quality control samples were measured at low, medium and high concentrations, i.e. 8.1 ng/mL, 60.8 ng/mL and 121.6 ng/mL abrin for within-/between-day precision and accuracy assessment. The common peptides YEPTVR, QFIEALR and EQQWALYTDGSIR were used for quantification of the total abrin content due to their 6

Figure 4. Standard curves of four exemplarily chosen peptides in milk. Abrin and agglutinin toxins were spiked in increasing concentrations into milk. After immunocapture and tryptic digest, resulting peptides were measured with the immunocapture LC-MS method. The added AQUA peptides allowed the generation of specific 8-point standard curves (dots, weighing 1/x). Five independently processed quality control samples at three levels (squares) verified the reliability of the method. Peptides of all isoforms and agglutinin were detected with high sensitivity and linearity (see also Supporting Information S4).

Furthermore, the stability of a toxin-containing sample is an important criterion for the usability of a method. Abrin-containing milk samples were processed directly after the spiking or incubated for five hours at room temperature. Both conditions resulted in comparable area ratios for YEPTVR, QFIEALR, and EQQWALYTDGSIR peptides demonstrating the stability of abrin under this condition (Table 1). Taking these results together, the PRM method was proven to be reliable for abrin quantification in milk.

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Table 1. Evaluation of immunocapture MS method in milk. Low concen- Medium concen- High concentratration tration tion (8.1 ng/mL) (60.8 ng/mL) (121.6 ng/mL) Accuracy [%] 84.5 – 110.4

107.2 – 118.0

98.5 – 112.1

CV%intra[%]

5.5 – 17.3

2.6 – 12.6

5.9 – 10.3

CV%inter [%]

8.4 – 14.8

7.8 – 9.5

8.2 – 10.6

91.8 – 98.4

81.7 – 88.9

Stability* [%] 78.1 – 88.7

Accuracy and CV% of concentration determination were evaluated for the three common peptides YEPTVR, QFIEALR and EQQWALYTDGSIR independently. All values are given as minimum and maximum percentage. *as expressed as ratio of measured concentrations after 5 hours of incubation to direct processing (T0).

Quantification of abrin isoforms in other matrices. To analyze the performance of the newly established assay, abrin was quantified in various foods, environmental and clinical matrices. Besides milk, which was used for the evaluation study, ham was selected as solid food matrix. As examples for environmental matrices, river water and soil were chosen, representing liquid and solid materials. Human plasma was analyzed as potential clinical matrix. Each matrix was spiked with increasing concentrations of purified abrin. After immunocapture and tryptic digest, resulting peptides were measured using the PRM method. Linearity was evaluated by the generation of a matrixspecific standard curve: The performance for four exemplarily chosen peptides, representing different isoforms, is shown in Figure S5 (Supporting Information). Moreover, quality control samples at three concentrations were employed to evaluate the within-day repeatability. Peptides of all isoforms and agglutinin were detected in all matrices above 3 ng/mL of corresponding protein. The resulting standard curves were highly linear (R² >0.97) and the slope for abrin peptides was comparable to the standard curves in milk (Figure S5, Supporting Information). The slope of the standard curve of the agglutinin peptide was more impacted by different matrices, probably by a higher influence of matrix effects on antibody-protein interactions (Figure S5, Supporting Information). The independently processed quality control samples (same concentrations as used for milk) proved the high repeatability of the assay with CV found below 15% for all evaluated concentrations. No peptides were detected in blank samples, so the method is highly specific. Here, the combination of enrichment by antibodies and the sequence confirmation by LCMS/MS led to a highly reliable method that even worked with toxin-spiked complex matrices. The examined matrices represent a broad spectrum of potential carrier materials that could be found in a biological threat scenario making the method generally attractive for rapid identification of abrin intoxication in expert laboratories.

CONCLUSION Taking all results together, the newly established method was proven to be reliable for rapid abrin quantification in complex foods, clinical or environmental matrices. To our knowledge, this is the first quantitative MS-based assay for abrin, constituting a complementary approach to currently available ELISA and lateral flow assays. The time-consuming digestion step, typically required for bottom-up MS-based assays, was efficiently shortened through tryptic digestion in a bath-type sonicator. The evaluation study showed reproducible and efficient

peptide release in the brief incubation time. Furthermore, sequence heterogeneity of the abrin proteins was considered during assay development. The multi-epitopes immunocapture step and the monitoring of multiple proteotypic peptides, common or isoform specific, were designed to avoid any loss of signal due to isoforms or potential protein variants. In addition, it provides the capability for unambiguous identification of different abrin isoforms and their precise differentiation, which constitutes a clear benefit over current immunological assays. In the future, the novel method can be used to gain insights into the relative proportion of abrin isoforms and agglutinin obtained from different A. precatorius cultivars originating from various geographical locations in a dedicated forensic-oriented study. The method is intended to be implemented in regulatory expert laboratories for confirmation of results obtained with rapid field detection assays. Depending on available instruments, peptides monitoring by targeted high resolution mass spectrometry could be adapted to the alternative SRM detection on a triple quadrupole instrument. Ultimately, the protocol could be extended to other highly toxic proteins, e.g. ricin.

ASSOCIATED CONTENT Supporting Information Additional information is noted in text. The Supporting Information in pdf format is available free of charge on the ACS Publications website.

AUTHOR INFORMATION Corresponding Author * Phone: +33-1-69-08-13-15. Fax: +33-1-69-08-59-07. E-mail: [email protected].

Notes The authors declare no competing financial interest.

Author Contributions All authors have given approval to the final version of the manuscript.

ACKNOWLEDGMENT This project was supported by the French joint ministerial program of R&D against CBRNE risks and by grants from the German Federal Ministry of Education and Research to BGD (GEFREASE project, 13N132223).

REFERENCES (1) Select Agents and Toxins https://www.selectagents.gov/SelectAgentsandToxinsList.html. (2) Dickers, K. J.; Bradberry, S. M.; Rice, P.; Griffiths, G. D.; Vale, J. A. Toxicol. Rev. 2003, 22, 137–142. (3) Jansen, H. J.; Breeveld, F. J.; Stijnis, C.; Grobusch, M. P. Clin. Microbiol. Infect. 2014, 20, 488-496. (4) Gill, D. M. Microbiol. Rev. 1982, 46, 86–94. (5) Balali-Mood, M.; Moshiri, M.; Etemad, L. Toxicon Off. J. Int. Soc. Toxinology 2013, 69, 131–142. (6) Audi, J.; Belson, M.; Patel, M.; Schier, J.; Osterloh, J. JAMA 2005, 294, 2342–2351. (7) Olsnes, S.; Refsnes, K.; Pihl, A. Nature 1974, 249, 627–631. (8) Olsnes, S.; Heiberg, R.; Pihl, A. Mol. Biol. Rep. 1973, 1, 15– 20. (9) Endo, Y.; Mitsui, K.; Motizuki, M.; Tsurugi, K. J. Biol. Chem. 1987, 262, 5908–5912. (10) Endo, Y.; Tsurugi, K. Nucleic Acids Symp. Ser. 1986, 187– 190. (11) Mishra, R.; Kumar, M. S.; Karande, A. A. Mol. Cell. Biochem. 2015, 403, 255–265.

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