David J. Bourne

Detection of intact ricin in crude and purified extracts from castor beans using matrix-assisted laser desorption ionization mass spectrometry.

Ricin is a highly toxic protein from the seeds of the castor bean plant. Crude extracts from castor beans are toxic by several routes, and there is international concern about the use of these extracts by terrorist organizations. Lethality in aerosolized form has spurred the development of methods for the rapid detection of this protein from air samples that is critical in determining the illicit use of this material. Matrix-assisted laser desorption ionization (MALDI) mass measurement with an automated laser firing sequence was used to detect intact ricin from solutions containing less than 4 microg/mL of ricin in the presence of other endogenous seed proteins. This sensitivity was attained with the addition of 0.01% Tween 80 to the extracts that greatly enhanced the ricin signal. Importantly, this treatment substantially reduces the interference from the castor bean seed storage proteins. Commonly the ricin signal can be completely obscured by the oligomers of seed storage proteins, and this treatment reveals the ricin molecular ion, allowing the analyst to make a judgment as to the ricin content of the extract. This method provides for sensitive and rapid identification of intact ricin from aqueous samples with little sample preparation and is amenable to automatic acquisition.

A Study of the Metabolome of Ricinus communis for Forensic Applications

Investigations were undertaken to ascertain the appropriateness of studying the metabolome of Ricinus communis for cultivar and provenance determination. Seeds from 14 R. communis specimens (a total of 56 seeds) collected from the east coast of Australia were analyzed by high pressure liquid chromatography with UV detection (HPLC-UV), liquid chromatography–mass spectrometry (LC-MS), and 1H NMR spectroscopy. The collected data were then analyzed using principle component analysis (PCA). For HPLC-UV analysis, six R. communis specimens were unambiguously identified by PCA as belonging to separate classes relating to specimen. LC-MS data allowed unique ions to be identified for four specimens. Conversely 10 specimens were unambiguously segregated in the PCA of the 1H NMR data. The ratio of ricinine 1 to demethylricinine analogues 2 and 3 was found to be important for specimen determination. These combined analyses suggested that a combination of HPLC-UV and 1H NMR in conjunction with PCA could allow for specimen differentiation.

Metabolomic investigations of Ricinus communis for cultivar and provenance determination

Eight specimens of six known cultivars of Ricinus communis were investigated for differences in their metabolome that could be used to determine both provenance and cultivar. Seven replicates of three seeds per specimen were subjected to 1H NMR analysis, with the collected data further investigated using multivariate statistical analysis (OPLS-DA), resulting in class separations according to provenance. Analysis of loadings plots in addition to further chemical analysis of the extracts allowed for the identification of phenylalanine, ricinine, the N-demethyl and O-demethyl analogues of ricinine, and sucrose as important molecular markers for particular cultivars. To test the strength of the model, extracts generated from blinded specimens were used as a prediction set and were correctly classified according to provenance and cultivar.

Comparative investigation of the interaction of Zn(II), Cd(II), Ag(I) and Pb(II) with dibenzo-substituted macrocyclic ligands incorporating both symmetrically and unsymmetrically arranged N, O and S donors: synthetic, solution and X-ray studies

A comparison of the interaction of Zn(II), Cd(II), Ag(I) and Pb(II) with a matrix of 18, 17-membered, dibenzo-substituted macrocyclic ligands (1–18) incorporating nitrogen, oxygen and/or sulfur heteroatom sites that share the same carbon backbone is reported. Potentiometric log K data for complexes of these metals with 5, 6, 8 and 9 incorporating unsymmetric arrangements of their donor sets were obtained and the results compared with previously determined stability data for the remaining symmetrical and unsymmetrical ligand complexes forming the above matrix. Structure–function relationships underlying the relative magnitudes of the respective log K values are discussed, with emphasis given to the influence of both the donor atom set and the donor atom sequence on the nature of the resulting complexes. The X-ray structures of [CdL(NO3)] (NO3)·CH3CH2OH (L = 4, SN4 donor set), [CdL(NO3)2]·CH3OH (L = 7, ON4 donor set), [AgL]PF6 (L = 4, SN4 donor set) and [AgL]PF6 (L = 9, OSN3 donor set) are presented along with that of the metal-free diprotonated nitrate salt (LH2)(NO3)2 (L = 6, S2N3 donor set).

Selected non-ionic biological detergents enhance signal intensity of intact bovine serum albumin by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry

Recently, we showed that the signal intensity of intact protein by matrix-assisted laser desorption/ionisation (MALDI) mass spectrometry measurement can be enhanced at least an order of magnitude by the addition of Tween80 to the analyte solution. We did not ascertain whether this effect was limited to Tween80 or if it was more universal of biological detergents. This paper discusses our investigations into this question. A variety of chemically diverse detergents were added to analyte solutions containing bovine serum albumin (BSA) to determine whether there was significant signal enhancement. The addition of Tween20, Tween80, Triton X-100 and Triton X-114 improved the attainable sensitivity of intact protein MALDI mass spectrometry compared to spectra acquired without detergent. In some cases there was considerable improvement in signal—for example, with Triton X-100 two charge states (the +1 and +2) of BSA (3.9 fmol) could easily be observed. Another advantage of this process is that the detergent can be added directly to the matrix solution reducing sample handling and preparation time. We propose this phenomenon results from the ability of these detergents to increase the solubility of the protein via hydrophobic and hydrophilic interactions between the detergent and protein. The increased solubility allows for more uniform deposition of the analyte/matrix mixtures producing an evenly distributed layer of analyte especially useful for data acquisition using an automated laser firing sequence.