Sten-Åke Fredriksson

A concept study on identification and attribution profiling of chemical threat agents using liquid chromatography–mass spectrometry applied to Amanita toxins in food

Accidental or deliberate poisoning of food is of great national and international concern. Detecting and identifying potentially toxic agents in food is challenging due to their large chemical diversity and the complexity range of food matrices. A methodology is presented whereby toxic agents are identified and further characterized using a two-step approach. First, generic screening is performed by LC/MS/MS to detect toxins based on a list of selected potential chemical threat agents (CTAs). After identifying the CTAs, a second LC/MS analysis is performed applying accurate mass determination and the generation of an attribution profile. To demonstrate the potential of the methodology, toxins from the mushrooms Amanita phalloides and Amanita virosa were analyzed. These mushrooms are known to produce cyclic peptide toxins, which can be grouped into amatoxins, phallotoxins and virotoxins, where α-amanitin and β-amanitin are regarded as the most potent. To represent a typical complex food sample, mushroom stews containing either A. phalloides or A. virosa were prepared. By combining the screening method with accurate mass analysis, the attribution profile for the identified toxins and related components in each stew was established and used to identify the mushroom species in question. In addition, the analytical data was consistent with the fact that the A. virosa specimens used in this study were of European origin. This adds an important piece of information that enables geographic attribution and strengthens the attribution profile.

Deamidation in ricin studied by capillary zone electrophoresis- and liquid chromatography-mass spectrometry.

Deamidation in ricin, a toxin present in castor beans from the plant Ricinus communis, was investigated using capillary zone electrophoresis (CZE) and liquid chromatography coupled to high resolution mass spectrometry. Potential sites for deamidation, converting asparagine (Asn) into aspartic or isoaspartic acid (Asp or isoAsp), were identified in silico based on the protein sequence motifs and tertiary structure. In parallel, CZE- and LC-MS-based screening were performed on the digested toxin to detect deamidated peptides. The use of CZE-MS was critical for the separation of small native/deamidated peptide pairs. Selected peptides were subjected to a detailed analysis by tandem mass spectrometry to verify the presence of deamidation and determine its exact position. In the ricin preparation studied, deamidation was confirmed and located to three asparagine residues: Asn54 in the A-chain, and Asn42 and Asn60 in the B-chain. Possible in vitro deamidation occurring during sample preparation was monitored using a synthetic peptide with a known and rapid rate of deamidation. Finally, we showed that the isoelectric diversity previously reported in ricin is related to the level of deamidation.

Identification of RIP-II toxins by affinity enrichment, enzymatic digestion and LC-MS.

Type 2 ribosome-inactivating protein toxins (RIP-II 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.

ABH blood group antigens in N-glycan of human glycophorin A

We previously showed that a small proportion of the O-linked oligosaccharide chains of human glycophorin A (GPA) contains blood group A, B or H antigens, relevant to the ABO phenotype of the donor. The structures of these minor O-glycans have been established (Podbielska et al. (2004) [20]). By the use of immunochemical methods we obtained results indicating that ABH blood group epitopes are also present in N-glycan of human GPA (Podbielska and Krotkiewski (2000) [22]). In the present paper we report a detailed analysis of GPA N-glycans using nanoflow electrospray ionization tandem mass spectrometry. N-glycans containing A-, B- and H-related sequences were identified in GPA preparations obtained from erythrocytes of blood group A, B and O donors, respectively. The ABH blood group epitopes are present on one antenna of the N-glycan, whereas a known sialylated sequence NeuAcalpha2-6Galbeta1-4GlcNAc- occurs on the other antenna and other details are in agreement with the known major structure of the GPA N-glycan. In the bulk of the biantennary sialylated N-glycans released from GPA preparations, the blood group ABH epitopes-containing N-glycans, similarly O-glycans, constituted only a minor part. The amount relative to other N-glycans was estimated to 2-6% of blood group H epitope-containing glycans released from GPA-O preparations and 1-2% of blood group A and B epitope-containing glycans, released from GPA-A and GPA-B, respectively.

Recommended Mass Spectrometry-Based Strategies to Identify Ricin-Containing Samples

Ricin is a protein toxin produced by the castor bean plant (Ricinus communis) together with a related protein known as R. communis agglutinin (RCA120). Mass spectrometric (MS) assays have the capacity to unambiguously identify ricin and to detect ricin’s activity in samples with complex matrices. These qualitative and quantitative assays enable detection and differentiation of ricin from the less toxic RCA120 through determination of the amino acid sequence of the protein in question, and active ricin can be monitored by MS as the release of adenine from the depurination of a nucleic acid substrate. In this work, we describe the application of MS-based methods to detect, differentiate and quantify ricin and RCA120 in nine blinded samples supplied as part of the EQuATox proficiency test. Overall, MS-based assays successfully identified all samples containing ricin or RCA120 with the exception of the sample spiked with the lowest concentration (0.414 ng/mL). In fact, mass spectrometry was the most successful method for differentiation of ricin and RCA120 based on amino acid determination. Mass spectrometric methods were also successful at ranking the functional activities of the samples, successfully yielding semi-quantitative results. These results indicate that MS-based assays are excellent techniques to detect, differentiate, and quantify ricin and RCA120 in complex matrices.

Acetylcholinesterases from the Disease Vectors Aedes aegypti and Anopheles gambiae: Functional Characterization and Comparisons with Vertebrate Orthologues.

Mosquitoes of the Anopheles (An.) and Aedes (Ae.) genus are principal vectors of human diseases including malaria, dengue and yellow fever. Insecticide-based vector control is an established and important way of preventing transmission of such infections. Currently used insecticides can efficiently control mosquito populations, but there are growing concerns about emerging resistance, off-target toxicity and their ability to alter ecosystems. A potential target for the development of insecticides with reduced off-target toxicity is the cholinergic enzyme acetylcholinesterase (AChE). Herein, we report cloning, baculoviral expression and functional characterization of the wild-type AChE genes (ace-1) from An. gambiae and Ae. aegypti, including a naturally occurring insecticide-resistant (G119S) mutant of An. gambiae. Using enzymatic digestion and liquid chromatography-tandem mass spectrometry we found that the secreted proteins were post-translationally modified. The Michaelis-Menten constants and turnover numbers of the mosquito enzymes were lower than those of the orthologous AChEs from Mus musculus and Homo sapiens. We also found that the G119S substitution reduced the turnover rate of substrates and the potency of selected covalent inhibitors. Furthermore, non-covalent inhibitors were less sensitive to the G119S substitution and differentiate the mosquito enzymes from corresponding vertebrate enzymes. Our findings indicate that it may be possible to develop selective non-covalent inhibitors that effectively target both the wild-type and insecticide resistant mutants of mosquito AChE.

A ricin forensic profiling approach based on a complex set of biomarkers

A forensic method for the retrospective determination of preparation methods used for illicit ricin toxin production was developed. The method was based on a complex set of biomarkers, including carbohydrates, fatty acids, seed storage proteins, in combination with data on ricin and Ricinus communis agglutinin. The analyses were performed on samples prepared from four castor bean plant (R. communis) cultivars by four different sample preparation methods (PM1-PM4) ranging from simple disintegration of the castor beans to multi-step preparation methods including different protein precipitation methods. Comprehensive analytical data was collected by use of a range of analytical methods and robust orthogonal partial least squares-discriminant analysis- models (OPLS-DA) were constructed based on the calibration set. By the use of a decision tree and two OPLS-DA models, the sample preparation methods of test set samples were determined. The model statistics of the two models were good and a 100% rate of correct predictions of the test set was achieved.