Identification of RIP-II Toxins by Affinity Enrichment, Enzymatic

Dec 12, 2014 - Good recoveries above 50% were obtained for water and samples of beverages such as beer and apple juice (Table 1). However, recoveries ...
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Identification of RIP-II Toxins by Affinity Enrichment, Enzymatic Digestion and LC-MS Sten-Åke Fredriksson,* Elisabet Artursson, Tomas Bergström, Anders Ö stin, Calle Nilsson, and Crister Åstot Swedish Defence Research Agency (FOI), CBRN Defence and Security, SE-901 82 Umeå, Sweden S Supporting Information *

ABSTRACT: Type 2 ribosome-inactivating protein toxins (RIPII toxins) were enriched and purified prior to enzymatic digestion and LC-MS analysis. The enrichment of the RIP-II family of plant proteins, such as ricin, abrin, viscumin, and volkensin was based on their affinity for galactosyl moieties. A macroporous chromatographic material was modified with a galactose-terminated substituent and packed into miniaturized columns that were used in a chromatographic system to achieve up to 1000-fold toxin enrichment. The galactose affinity of the RIP-II proteins enabled their selective enrichment from water, beverages, and extracts of powder and wipe samples. The enriched fractions were digested with trypsin and RIP-II peptides were identified based on accurate mass LC-MS data. Their identities were unambiguously confirmed by LC-MS/MS product ion scans of peptides unique to each of the toxins. The LC-MS detection limit achieved for ricin target peptides was 10 amol and the corresponding detection limit for the full method was 10 fmol/mL (0.6 ng/mL). The affinity enrichment method was applied to samples from a forensic investigation into a case involving the illegal production of ricin and abrin toxins.

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in accordance with the CWC. However, the plant is very decorative and its widespread use in gardening and public park plantations makes the seeds readily available. Previous uses of ricin and concerns regarding its relevance in antagonistic threats have highlighted the need for methods to allow its detection and chemical analysis. In addition to the assassination of the Bulgarian dissident Markov in London 1978 and a similar attack on Vladimir Kostov in Paris during the same year, ricin has been used in numerous threat letter incidents (powder letters), murders, and suicides.6 The toxicity of RIP-II toxins depends strongly on the route of administration. The LD50 values reported for ricin and abrin are in the range 0.2−10 μg/kg body weight for inhalation and injection. The toxicity values determined for viscumin and modeccin were in the same range,7,8 but the agglutinins RCA and Abrus precatorius agglutinin (APA) were 2−3 orders of magnitude less toxic.7,9 The toxicity of RIP-II toxins by ingestion was reported to be 3 orders of magnitude lower than by injection or inhalation.10 Immunoassays and functional tests are available for the determination of ricin and other RIP-II toxins. In functional assays such as the luciferase luminescence test11 the enzymatic activity of the A-chain is determined using a cell lysate. Other

ype 2 ribosome-inactivating proteins (RIP-II toxins) are a class of plant toxins that includes a number of potent chem-bio threat agents, such as ricin (Ricinus communis), abrin (Abrus precatorius), viscumin (Viscum album), modeccin (Adenia digitata), and volkensin (Adenia volkensii). RIP-II toxins are heterodimeric proteins that consist of an enzymatically active N-glycosidase A chain connected to a B chain via a disulfide bond. The B chain is a lectin with carbohydrate binding domains that have an affinity for galactose-terminated surface receptors on eukaryotic cells. Binding promotes the uptake of the toxin into the cell by endocytosis, where a part intriguingly finds its way via the Golgi complex to the endoplasmatic reticulum. From there the A subunit is translocated into the cytosol, where it hydrolyses the Nglycosidic bond between an adenine residue and ribose at a specific position in 28S rRNA and thereby inhibits protein synthesis, eventually causing cell death.1,2 Among the RIP-II toxins, ricin is of primary concern because it has been included in chem-bio weapon programs.3 Since 1997, its production and possession have been regulated by the Chemical Weapons Convention (CWC).4 Ricin is found in the castor bean plant, Ricinus communis, together with the very similar toxin Ricinus communis agglutinin (RCA). Ricinus communis is cultivated on a large scale and more than 2 million metric tons of seeds are currently harvested per annum for the industrial production of castor oil.5 Ricin is destroyed during standard oil production processes, usually by heat inactivation, © 2014 American Chemical Society

Received: September 2, 2014 Accepted: December 12, 2014 Published: December 12, 2014 967

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previously published procedures.22 Ricinus communis agglutinin (RCA120), viscumin (Viscum album), peanut agglutinin (PNA, Arachis hypogaea), and bovine serum albumin (BSA) were obtained from Sigma. Crude extracts were prepared in house from various cultivars of Ricinus communis castor beans and Abrus precatorius seeds obtained from a local market in Egypt.22,32 Poros 20 AL aldehyde activated affinity medium was obtained from Applied Biosystems. Millex HA membrane filters were obtained from Merck Millipore and Nanosep MF GHP centrifugal filters from Pall Corporation. The ligand paminophenyl-1-thio-β-D-galactopyranoside was bought from Bachem AG, Switzerland. N-Hydroxysuccinimide (NHS) and 1-ethyl-3-(3-(dimethylamino)propyl) carbodiimide (EDC) were acquired from Sigma-Aldrich. Ammonium acetate, methanol, acetonitrile, and trifluoroactetic acid (TFA) were of analytical or LC-MS grade. Water was obtained from a MilliQ system. Buffers in the pH interval 0.8−7.3 were prepared from citric acid, acetic acid, potassium hydrogen phosphate and tris(hydroxymethyl)aminomethane (Tris). Sequencing grade modified trypsin was obtained from Promega. Toxin Samples and Sample Preparation. Crude Extracts. Crude extracts of ricin and abrin were prepared as described previously.22 Briefly, 0.5−1 g of seeds were homogenized in 4 mL of diethyl ether and transferred to a screw-capped polypropylene test tube. Four milliliters of 0.5% acetic acid was then added and the suspension was mixed for 30 s on a vortex mixer. After centrifugation, the aqueous phase was recovered and filtered through a 0.45 μm Millex HA syringe filter. The resulting extract was stored at 4 °C. Wipe Samples. Simulated ricin powder samples were prepared by distributing 10 μL of a ricin extract containing 1 μg ricin on a 1 cm2 glass surface in a Petri dish. After evaporation, the dried spot was sampled by wiping with a Dacron-tipped swab (New Horizons Diagnostics Corp., USA) wetted with PBS containing 0.1 mg of bovine serum albumin (BSA)/mL. The tip of the swab was cut into a microcentrifuge tube and vortexed for 20 s in 1 mL of PBS containing 0.1 mg BSA/mL. The extract was filtered using a 0.2 μm Nanosep MF GHP centrifugal filter. Beverages. Simulated threat samples for method evaluation were prepared by spiking drinking water and various locally purchased beverages with a crude extract of ricin that also contained RCA. The samples were spiked at approximately 1 μg ricin/mL. Affinity Sample Purification and Enrichment. The affinity chromatography packing material was prepared by coupling a galactose-terminated ligand to Poros AL chromatographic resin according to the manufacturer’s instructions. Briefly, 100 mg of Poros AL, 300 μL of 20 mM 4-aminophenyl1-thio-β-D-galactopyranoside ligand solution, and 140 μL of 1.5 M Na2SO4 in 100 mM pH 7.4 phosphate buffer were mixed in a screw-capped microcentrifuge tube. Solid sodium cyanoborohydride (1.9 mg) was added and the mixture was agitated for 90 min to allow the coupling reaction to proceed. Unreacted aldehyde groups were capped by adding 600 μL of 0.2 M Tris containing 5 mg/mL of sodium cyanoborohydride. After capping, the resin was washed with 150 mM NaCl in 10 mM pH 7.4 phosphate buffer. Poros affinity material was prepared using ligand concentrations in the 1−20 mM range and the capacity of the resulting media was evaluated using purified ricin D. In addition, cyanogen bromide-activated Sepharose affinity material with

functional tests assess the enzymatic activity of the A chain against RNA by using mass spectrometry to quantify the hydrolysis of adenine in a substrate molecule that is added to the sample.12,13 Alternatively, cell-based cytotoxicity tests that assess the toxin’s full functionality are available.14 These tests are highly sensitive and can detect toxin activity at concentrations of less than 1 ng/mL and immunoassays such as ELISA using specific antibodies have demonstrated similar sensitivity.15−18 It is often required to use mass spectrometric techniques to unambiguous identify target substances in conformity with relevant identification criteria, such as those defined by the Organisation for the Prohibition of Chemical Weapons (OPCW) for verification of the CWC,19 by the EU for analysis of contaminants in food,20 and by WADA for doping analysis.21 Samples to be analyzed by MS for the determination of protein toxins present at low concentrations often require cleanup and enrichment to achieve adequate sensitivity. This can be done in a variety of ways. Nonspecific isolation of toxins using molecular weight filtration has been used for less complex samples.22,23 Immunoaffinity-based cleanup and enrichment methods have been developed for the determination of ricin in more complex matrices.24−26 Notably, a protocol involving immunological cleanup followed by trypsin digestion and MALDI MS analysis of the digested products has been used to determine ricin levels in crude ricin extracts and milk samples. In addition, a multiplexed immunoaffinity cleanup protocol for the analysis of ricin, staphylococcus enterotoxin B (SEB) and botulinum neurotoxins A and B in milk and juice samples was described in which a mixture of paramagnetic beads with immobilized monoclonal antibodies for the different toxins is used in a specific MALDI mass spectrometric screening assay.27 For RIP-II toxins enrichment the use of galactose as the affinity ligand has the advantage over immunoaffinity that the ligand can be used to enrich the entire family of RIP-II toxins in a single step. Immunological sample preparation requires the availability of information on the target compound or the use of an array of different antibodies. Monoclonal antibodies are relatively expensive and are not commercially available for all RIP-II toxins. The modification of Sepharose chromatographic material with galactose as the affinity ligand for purification of ricin was originally described by Simmons and Russell.28 Lactosemodified Sepharose has been used for purification of abrin.29 Recently, paramagnetic nanoparticles modified with galactose and lactose-modified monolithic silica were both used for the affinity enrichment of ricin.30,31 The present work describes the coupling of a galactose ligand to a commercially available highly efficient chromatographic material for the use in a miniaturized system for high throughput trace enrichment of RIP-II toxins in water, beverages and wipe and powder sample extracts. The aim was to develop a general RIP-II preparation method for verification purposes and furthermore, the conditions for enzymatic digestion of the enriched toxin fraction were optimized and accurate mass LC-MS was used to detect predicted trypsin digest peptides of the RIP-II toxins ricin, abrin, and viscumin, their isoforms and the associated agglutinins.

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EXPERIMENTAL SECTION Chemicals and Materials. Ricin (RCA60) was purified from Ricinus communis castor beans of the zanzibariensis cultivar (Sandeman Seeds, United Kingdom) according to 968

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was reduced to 400 nL/min, the trapping valve was switched and the peptides were separated on a 150 mm, 75 μm i.d. BEH C18 nanoAcquity UPLC column (Waters) using a 20 min linear gradient from 3 to 40% acetonitrile containing 0.1% formic acid. The gradient was then stepped to 80% acetonitrile, which was held for 4 min before reverting to the initial conditions. LC-MS Analysis of the Nonbinding Fraction. A 5 mm 300 μm i.d. PepMap C18 trap (LC-Packings/Dionex) was used to analyze the nonretained fraction. A 5 μL aliquot was injected and the column was eluted using a 2 min gradient from 30% to 80% acetonitrile in water containing 0.1% formic acid at 7 μL/ min. The standard electrospray source was used and spectra were acquired over the mass range 300−2200. RIP-II Identification and Quantification. Ricin was identified and quantified by accurate mass LC-MS analysis of a ricin reference standard digested with trypsin. The concentration of the standard was determined by UV absorption measurement at 280 nm using the extinction coefficient 1.18 (mg/mL)−1 × cm−1.34 The responses of selected peptides relative to the leucine enkephalin internal standard were determined from extracted ion chromatograms (EICs) covering a 20 ppm mass window using the TargetLynx program (Waters). Provisional identifications of other RIP-II toxins were based on the predicted masses of their trypsin digest peptides, which were calculated from available database sequences.35 The EICs of doubly and triply charged molecular ions of medium sized peptides containing 6−20 amino acids were generated and the molecular weights deduced from the spectra of candidate peaks were compared to the corresponding theoretical values. Peptides containing cysteine and the Nglycosylation consensus sequence were not used for screening. The identifications were subsequently confirmed by comparing the retention times and product ion spectra for the samples under investigation to those observed in analyses of reference digests of RIP-II toxins stored at −80 °C. The concentrations of RIP-II toxins other than ricin were determined using a label free quantification method (“Hi3” or “Top 3 Protein Quantification”).36,37 The samples were quantified by comparing the averaged response of three high intensity peptides from each of the toxins to those of a ricin standard. Trypsin digest peptides specific to ricin and common to the D and E isoforms were selected (T7a, T11a, T6b) and a calibration curve was prepared using data from reference digests of purified ricin D. For the quantification of RCA the proteotypic peptides T6a, T9a, and T4b were used (Supporting Information, Tables 1−4.). Safety. RIP II toxins are very dangerous and should be handled only by appropriately trained personnel. Ricin is listed in the Chemical Weapons Convention and is thus subject to national and international regulations that must be strictly followed. Crude extracts of herbal material were prepared in a fume hood. Used equipment was decontaminated by immersion in 2 M sodium hydroxide for 2 h.

six-carbon spacer moieties was prepared by coupling 6aminohexanoic acid to the material before modifying it with the galactosyl ligand. The spacers were added using a standard EDC/NHS protocol.33 The effect of using zero, one, or two spacers on the capacity of the material was evaluated using purified ricin D. Miniaturized affinity columns were constructed from PEEK 10−32 to 1/4−28 adapters drilled to 1.6 mm internal diameter and terminated by a 2 μm outlet frit (Upchurch Scientific, WA, USA). The columns were slurry-packed in methanol at a flow rate of 2 mL/min and a Sure-Guard 2 μm in-line filter (VICI Jour, Switzerland) was attached to the inlet end, resulting in a 10 mm bed length. A simple chromatographic system consisting of a BioRad Duo-Flow chromatography system (Bio Rad Laboratories AB, Sweden), a Valco C6W injection valve (VICI AG International, Switzerland), an UV detector and a fraction collector was used to process the samples (Supporting Information, Figure 1). The signal from the UV detector was used to trigger the collection of the enriched RIP-II toxin fraction. RIP-II toxin enrichment was evaluated using a crude castor bean extract containing ricin and RCA. Sample loading was performed using a 50 mM ammonium acetate pH 6.2 buffer solution containing 50% methanol at a flow rate of 0.4 mL/min, which could be increased to 4 mL/ min when loading large volume samples. The RIP II fraction was desorbed from the column using 40 μL of 50 mM TFA in 50% methanol. Capillary Gel Electrophoresis. Intact proteins in the binding and nonbinding fractions were analyzed by capillary gel electrophoresis using a Bio-Rad Experion system. The concentration of RIP-II toxins was determined using the quantification protocol supplied by the instrument manufacturer. Enzymatic Digestion. The affinity enriched RIP-II fraction was evaporated to dryness at 70 °C under a mild nitrogen flow. Forty microliters of freshly prepared 100 mM ammonium bicarbonate (pH 7.8) and 2 μL of 0.2 μg/μL sequencing grade modified trypsin was added and the samples were digested at 40 °C for 2 h. Liquid Chromatography−Mass Spectrometry. The trypsin digests were analyzed on a nanoAcquity UPLC system interfaced to a Qtof Ultima mass spectrometer equipped with a Nano-Lockspray electrospray ion source (Waters Corporation, Milford, USA). Picotip fused silica spray tips (New Objective, MS Wil GmbH, Switzerland) were used. The TOF analyzer was operated at a resolution of 10000 (fwhm) and the MCP detector was integrated at 1 s intervals over the mass range 300−1800 in the MS-mode and 100−1800 in the MSMS mode. The mass scale was calibrated using product ions from Glufibrinopeptide B. Lock mass correction for drift and short-term instability was applied using the doubly charged molecular ion of Glu-fibrinopeptide B at m/z 785.8421. A 100 fmol/μL solution was introduced at 100 nL/min using the auxiliary pump of the nano-LC system. The reference sprayer orthogonal to the analyte sprayer was selected during 0.5 s every 30 s by switching a rotating baffle. The digests were diluted to 50−1000 μL with 0.4% formic acid containing leucine enkephalin as an internal standard. Depending on the expected amount of protein in the sample, 1−10 μL of the digest was injected and trapped on a 20 mm 180 μm i.d. C18 trap column (5 μm Symmetry C18, Waters) using 15 μL/min of 0.1% formic acid. After 1.5 min the flow

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RESULTS AND DISCUSSION Affinity Enrichment. RIP-II toxins are lectins with an affinity for the β-1,4-linked galactose found in mammalian cell surface glycolipids and glycoproteins. Carbohydrate binding has been utilized for selective extraction of RIP-II toxins from plant material as the first step in large scale purification procedures. While preparatory scale purifications often rely on Sepharose affinity material,29,38,39 the aim of this work was to develop 969

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An extra benefit of using a UV detector to monitor and control fraction collection was that high toxin concentration samples would be detected and the resulting RIP-II fractions could be diluted to avoid overloading the LC-MS instrument with excessively high levels of peptides. High UV absorption also indicated a need to replace the affinity column to avoid cross-contamination of subsequent low concentration samples. The speed and repeatability of the affinity separation was demonstrated by repeatedly injecting a sequence of one blank sample followed by four 100 μL crude ricin samples (Figure 2A). After elution of the nonbinding fraction, 40 μL of

miniaturized affinity columns suitable for trace enrichment and high sample throughput. Pressure-tolerant Poros perfusion material was therefore evaluated as the stationary phase. The material consists of 20 μm particles that are readily packed into columns and have large flow-through pores that enable rapid mass transfer without sacrificing efficiency at high flow rates. The galactosyl ligand was coupled directly to aldehyde groups on the chromatographic support material. Experiments using spacer-modified Sepharose beads demonstrated that steric effects had no influence on the affinity of ricin as compared to galactosyl-functionalized column materials with no spacer moieties. This observation is consistent with the readily accessible location of the carbohydrate binding sites of ricin and other RIP-II toxins on the surface of chain B.40 Measurements of the modified materials’ capacities indicated that the optimum surface density was achieved by functionalizing the stationary phase using ligand concentrations above 10 mM (data not shown). Using purified ricin, the capacity of the galactosylmodified Poros material was determined to be approximately 1 mg of RIP-II toxin/ml of packing material. For preparative purification of RIP-II toxins, the sample is normally desorbed using a high concentration of galactose or lactose. However, lactose did not completely elute the adsorbed lectins from the Poros material (Supporting Information, Figure 2). The effect of pH on the column material’s affinity for ricin was evaluated by loading a castor bean extract containing 10 μg of a mixture of ricin and RCA onto the column. The desorption efficiencies of a series of buffers with pH values ranging from 0.8−7.3 were evaluated and the column retained a high affinity for ricin and RCA at pH values above 5 and for quantitative desorption a buffer at pH 2 or less was required (Figure 1).

Figure 2. UV chromatograms of the trace enrichment of water samples spiked with 10 μg of crude ricin. (A) Repeated 100 μL injections of a sequence of one blank sample followed by 4 samples at 100 μg/mL concentration. (B) Two 10 mL injections of a 1 μg/mL sample. Blue arrows: sample injection. Red arrows: Peak from the RIP-II fraction.

desorption buffer was injected and the RIP-II fraction was collected. At a flow rate of 1 mL/min, almost 60 samples per hour could be processed using manual injection. For trace enrichment of large volume samples, the flow rate was increased to 3 mL/min. The recovery was independent of sample volume and flow rate as observed by the almost identical peak heights for the retained fractions from 100 μL and 10 mL samples, both containing 10 μg of ricin (Figure 2). The galactose affinity fractionation method was further evaluated using crude extracts of ricin and abrin, both also containing the corresponding agglutinin, and standard solutions of viscumin and peanut agglutinin (PNA). The fractions were evaluated by capillary gel electrophoresis. The crude castor bean extract yielded bands at 13, 67−70, and 144 kDa, corresponding to 2S-albumin, ricin and RCA, respectively (Figure 3, lane 1). 2S-Albumin was not retained on the affinity column but ricin and RCA interacted with the galactose ligand and were found in the retained fraction. Abrin and viscumin

Figure 1. Effect of pH on desorption of RIP-II toxins from the affinity column.

This observation is consistent with the reported pH dependency of ricin’s affinity for galactosyl moieties, which was attributed to a change in the protein’s three-dimensional structure from a compact globular form to a more extended denatured conformation.41 A pH 6 ammonium acetate buffer was used for sample loading, after which quantitative desorption was achieved using 40 μL of 50 mM TFA. All buffers contained 50% of methanol to reduce secondary interactions and assist the subsequent enzymatic digestion of the proteins. The miniaturized column format allowed the RIPII toxins to be obtained in a fraction volume of 100 μL, which facilitated further sample processing. The affinity columns were determined to have a ricin capacity of 20 μg, which was considered sufficient even for high concentration samples. 970

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large parts of their amino acid sequences in common. For example, 9 of the 30 predicted peptides (>5 aa) of ricin isoform D are common to RCA, and another 6 are common to ricin E (Supporting Information, Tables 1−4). Abrin exhibits strong sequence similarity to A. precatorius agglutinin-1 (APA), but only 2 trypsin digest peptides longer than 5 aa are common to any of the abrin isoforms a−d (Supporting Information, Tables 5−9). The digests of ricin samples diluted from an affinity-purified standard were analyzed by LC-MS and screened for peptides using EICs with a 20 ppm mass window (Figure 4). Figure 3. Capillary PAGE separation of lectin samples and the corresponding fractions from the affinity column: Bands at 1.2 and 280 kDa are molecular weight internal standards used by the software for alignment and quantification.

were also found in the retained fraction (60−70 kDa) but PNA appeared mainly in the nonbinding fraction (30 kDa). Ricin, RCA, abrin, and viscumin are lectins with Galβ1−4 specificity. Conversely, PNA has an affinity for Galβ1−3GalNActerminated carbohydrate structures. The weak retention of PNA on the galactose-functionalized column suggests that the specificity of its carbohydrate-binding domain is relatively high. Enzymatic Digestion. RIP-II toxins are resistant to proteolysis. The use of a denaturing agent in conjunction with reduction and alkylation has been recommended for ricin digestion,22 but alternative protocols for methanol-assisted rapid digestion of ricin and abrin have also been proposed.30 In addition, the beneficial effects of acid-labile surfactants on ricin digestion have been demonstrated.42 Initially, the methanolassisted approach for trypsin digestion was evaluated to avoid the addition of chaotrophic salts and surfactants as they may have adverse effects on the performance of the nano-LC system. The yield of peptides obtained by trypsin digestion of the acidic methanol-containing RIP-II fraction was variable and sometimes quite low (data not shown). This was attributed to the formation of a protein precipitate that was not accessible to trypsin. When the effect of methanol on the yield of digest peptides was investigated, it was found that after evaporation of the acidic methanol-containing RIP-II fraction, an aqueous digestion buffer was sufficient to obtain a high and consistent yield of peptides. These modified conditions reduced the amount of trypsin required for digestion, thereby minimizing background signals due to trypsin autolysis products and improved the analysis of trace concentration samples. The modified digestion protocol produced a consistent intensity of digest peptides over the working range of the Qtof instrument. The robust and efficient digestion achieved under these conditions implies that the low pH and the presence of methanol in the RIP-II fraction during its evaporation denatured the RIP-II proteins, which is consistent with the conformational changes reported for ricin at low pH.39 LC-MS Accurate Mass Monitoring and Identification. RIP-II toxins were provisionally identified and quantified using accurate mass extracted ion chromatograms (EICs) of their predicted trypsin digest peptides. The uniqueness of ricin trypsin digest peptides has been discussed previously.22 In general, peptides with more than 5−6 amino acids are often unique and not found in other proteins. However, RIP-II toxins and the associated agglutinins from the same plant often have

Figure 4. LC-MS response of 0.005−47 fmol of ricin trypsin digest peptides from a stepwise diluted crude castor bean extract. The inset shows an expansion of the low concentration samples corresponding to 5−150 amol of ricin on column. (Filled circles: Ricin-specific peptides. Open circles: Shared ricin/RCA peptides. Triangles: RCAspecific peptides.)

The responses of trypsin digest peptides suitable for ricin identification differed by a factor of 10. The most sensitive digest products suitable for identification were the peptides T10a and T11a, and the least sensitive was the glycopeptide T11b. The strongest overall response was achieved for T6a, which is common to ricin and RCA (HEIPVLPNR, T6a in ricin and T5a in RCA). Although not unique to ricin, the low detection limit of this peptide makes it an important marker for castor bean preparations. Analysis of Spiked Samples. The recovery of ricin and RCA from different sample matrices was evaluated by analyzing liquid samples and simulated powder samples spiked with 10 μL of an R. communis impala extract containing approximately 1 μg of ricin. The samples were purified by affinity enrichment prior to digestion and LC-MS analysis and compared with the result from same amount of extract injected as such on the affinity column. Good recoveries above 50% were obtained for water and samples of beverages such as beer and apple juice (Table 1). However, recoveries were significantly lower for beverages that are rich in carbohydrates and organic acids such as orange juice and certain soft drinks. For wipe sampling the nature of the sampling material was found to be critical. The best results were obtained with a Dacron-tipped wipe. Losses of RIP-II toxins during the handling of samples at the 100 μg/mL level were found to be insignificant. The recovery of ricin and RCA during affinity fractionation of the standard was determined to be 90% and 106%, respectively, relative to the standard digested without fractionation (Table 1). However, at trace levels nonspecific binding of the proteins 971

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similar behavior of the toxins in that matrix as well as the response of selected peptides. The accuracy has been shown to be in the order of 10−20% for protein standards added to complex mixtures.35,36 Thus, this procedure was accepted as adequate for the purpose of the analytical method used in this paper. The amount of RCA relative to ricin in the extract of the carmencita cultivar in Figure 4 was determined to 0.30 using the selected proteotypic RCA and ricin peptides. This value was validated by capillary gel electrophoresis analysis and corresponded well to the 0.33 ratio obtained (5% RSD, n = 3). Application to a Forensic Investigation. The new method was used to support a forensic investigation into a 32-year old man who was suspected of preparing toxic materials. On the basis of notes concerning toxin preparation found at the site, samples taken from the suspect’s home were submitted to the Swedish Defense Research Agency for characterization. The samples consisted of three glass bottles containing approximately 0.1 mL each of a cloudy liquid. Subsamples taken for screening using a functional protein synthesis assay11 exhibited strong RIP-II activity. An aliquot of 10 μL was therefore considered sufficient for affinity fractionation and LC-MS analysis. The nonretained fraction was analyzed directly by LC-MS and the retained RIP-II fraction was digested with trypsin before LC-MS analysis. Diagnostic Ricinus communis biomarker peptides44 were detected in the nonretained samples, albeit at low levels given the samples’ high activity in the functional assay (Supporting Information, Figure 3). Accurate mass EICs of trypsin digest peptides in the RIP-II fraction indicated that the samples contained moderate amounts of ricin and RCA along with a complex pattern of high intensity peptides (Figure 5). EICs for the expected trypsin digest peptides from other RIP-II toxins were therefore generated from the LC-MS data file, revealing a match between the major peptides and theoretical trypsin digest peptides of different abrin isoforms and the corresponding agglutinin (APA). Diagnostic peptides of ricin and abrin are highlighted in the LC-MS chromatogram shown in Figure 5. The general RIPII specificity of the affinity ligand enrichment allowed us to identify the major toxic components of the samples. The use of

Table 1. Recovery and Reproducibility in Samples Spiked with 1 μg of Ricin/mL Beverage and 0.3 μg of RCA/mL (1 mL Sample Volume, n = 3) ricin

RCA

sample

recovery (%)

%RSD

recovery (%)

%RSD

Standarda Tap water Mineral water Beer Apple juice Orange soft drink Wipe sampling from glass

90 70 65 52 52 40 45

15 21 14 7 6 19 10

106 64 68 40 46 28 47

23 24 12 9 5 13 4

a 10 μL of a R. communis extract in PBS and 0.1 mg BSA/mL containing 1 μg of ricin and 0.3 μg of RCA.

to sample containers and equipment was significant. To minimize such losses, BSA and sodium chloride were added to the samples at 0.1 mg/mL and 0.2 M, respectively. Although the sensitivity of the LC-MS instrument allowed detection of low attomol quantities of the target peptides, the method’s detection limit was largely determined by the recovery at trace concentrations. In water samples, a detection limit of 1−10 fmol of ricin/mL of sample was obtained. In the recovery experiments, ricin was quantified based on the responses of six ricin-specific peptides derived from both chains while RCA was quantified using three peptides. The relative standard deviations of the results obtained using the full method at 1 μg/mL ranged from 5 to 24% (Table 1), with the LC-MS analysis contributing 4−10% RSD. For accurate quantification the recovery in the expected concentration range in a particular matrix should be determined, either by spiking a known amount of the toxin to the sample, or using matrix matched calibration standards as has been suggested previously.43 In a forensic application (next section), the quantification method for ricin was validated with matrix-matched calibration standards in order to mimic the sample composition and reduce systematic errors. The accuracy was determined to 105% with a precision of 4% RSD (n = 3). The accuracy of the Top 3 quantification of other RIP-II toxins against the response of ricin peptides depends on a

Figure 5. LC-MS chromatogram of a trypsin digest of the RIP-II fraction from a sample containing ricin and abrin. Base peak intensity chromatogram (black) and EIC’s of peptides from ricin (red), abrin-a, -b, -c, and -d shared peptides (orange), abrin-a (blue), abrin-b, -c, -d (green), A. precatorius agglutinin (gray). 972

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Article

Analytical Chemistry

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an immunoaffinity based method for ricin enrichment, that could have been a natural choice after a positive functional test for ricin, might have resulted in the wrong assumption that ricin was the only threat agent at hand. To fulfill the requirements for unambiguous identification the samples were reanalyzed by LC-MS/MS together with reference digests of ricin and abrin. The retention times of the peptides in the sample, as well as their product ion spectra, were in good agreement with those of the reference digest (within ±0.2 min and minimum four product ions). Together with the positive results of the functional assay, this provided conclusive evidence for the presence of both toxins in the sample (Supporting Information, Table 10). The concentration of ricin in the sample whose chromatogram is shown in Figure 5 was determined to be 0.14 mg/mL by comparison with matrix-matched samples containing ricin D standard in an extract of Abrus precatorius. The concentration of abrin was estimated to be 1.6 mg/mL by comparing the averaged response of three high response peptides common to the abrin isoforms to the ricin calibration curve (Supporting Information, Tables 6−9). The concentration of RCA and APA were similarly estimated at 0.04 and 1.3 mg/mL, respectively. Thus, it can be concluded that the ribosome inactivation detected in the functional assay was primarily due to the presence of abrin rather than ricin.

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CONCLUSIONS A highly efficient chromatographic material was modified with a galactosyl ligand and successfully used for trace enrichment of RIP-II toxins. This affinity ligand enrichment was combined with accurate mass LC-MS analysis to provide evidence of intact, functional RIP-II toxins. The method was shown to have the capacity to enrich trace amounts of RIP-II toxins from various beverages and liquid extracts. The benefit of generic enrichment of RIP-II toxins was demonstrated in the analysis of samples from a forensic investigation, in which the method was used to identify abrin and ricin in mixed samples. Together with functional assay results, the LC-MS data provided unambiguous identification of ricin and abrin in the samples.

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ASSOCIATED CONTENT

S Supporting Information *

Additional information as noted in the text. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +46 90 106712. Fax: +46 90 106800. E-mail: sten-ake. [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Financial support from the Swedish Civil Contingencies Agency (MSB) and the Ministry of Defence is gratefully acknowledged.

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REFERENCES

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DOI: 10.1021/ac5032918 Anal. Chem. 2015, 87, 967−974

Article

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DOI: 10.1021/ac5032918 Anal. Chem. 2015, 87, 967−974