Liquid chromatography-mass spectrometry and chemometric analysis of Ricinus communis extracts for cultivar identification.

INTRODUCTION: Seeds of Ricinus communis contain the toxic protein ricin, a 64 kD heterodimeric type II ribosome-inactivating protein that has been used in several high-profile poisoning incidents. The ability to determine which cultivar the toxin was isolated from via an LC-MS method would be of significant use to law enforcement and forensic agencies. OBJECTIVE: To analyse via LC-MS and chemometrics (principal components analysis (PCA), orthogonal partial-least-squares discriminant analysis (OPLS-DA)) extracts of R. communis to identify compounds specific to a particular cultivar. METHODS: Seeds from eight specimens of six cultivars of R. communis ('carmencita', 'dehradun', 'gibsonii', 'impala', 'sanguineus' and 'zanzibariensis') were extracted using a standard methodology. These extracts were analysed by LC-MS then subjected to chemometric analysis (PCA and OPLS-DA). Identified compounds of importance were subjected to high-resolution Fourier transform (HRFT) MS and MS/MS to elucidate their structures. RESULTS: This analysis identified 17 ions as potential cultivar determinators. Through accurate mass measurement and MS/MS, molecular formulae for 13 ions were determined, including two known and 11 new peptides. CONCLUSION: Unique ions in extracts of 'carmencita', 'dehradun', 'gibsonii', 'impala' and 'zanzibariensis' were identified that would allow an individual cultivar to be distinguished from other cultivars in this study. Although 'sanguineus' extracts contained no unique compounds, a unique LC-MS profile would allow for cultivar assignment.

De novo sequencing of RCB-1 to -3: peptide biomarkers from the castor bean plant Ricinus communis.

Ricinus communis (also know as the castor bean plant) whose forbears escaped from suburban gardens or commercial cultivation grow wild in many countries. In temperate and tropical climates seeds will develop to maturity, and plants may be perennial. In Australia these plants have become widespread and are regarded as noxious weeds in many localities. The seeds of R. communis contain ricin, a protein toxin which can easily be extracted into an aqueous solution. Ricin is toxic by ingestion, inhalation, and injection. The history of terrorist and anarchist interest in the use of seeds from R. communis has driven the development of strategies for determination of cultivar and geographic location of the source of an extract of wild-grown castor bean seed. This forensic information is of considerable interest to law enforcement and intelligence organizations. During forensic studies of both the metabolome and proteome of extracts from eight specimens of six different cultivars of R. communis ("zanzibariensis" collected from Kenya and Tanzania, "gibsonii", "impala", "dehradun", "carmencita", and "sanguineus" collected from Spain and Tanzania), three peptide biomarkers (designated Ricinus communis biomarkers, or RCB) were identified in both the MALDI and electrospray LC-MS spectra. Two of these peptides (RCB-1 and RCB-2) were present in varying amounts in all cultivars, while RCB-3 was present only in the "carmencita" cultivar. The amino acid sequences of RCB-1 to -3 were determined using LC-MS(n) fragmentation and de novo sequencing on both the intact and the carbamidomethyl modified peptides. The connectivity of the two disulfide bonds that were present in all three RCB were determined using a strategy of partial reduction and differential alkylation using tris-(2-carboxyethyl)phosphine with N-ethylmaleimide to reduce and alkylate the most accessible disulfide bond, followed by reduction and alkylation of the remaining disulfide bond with dithiolthreitol and iodoacetamide. The possible functional role of RCB-1 to -3 in R. communis seeds is also discussed.

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.

The effect of Tween80 on signal intensity of intact proteins by MALDI time-of-flight mass spectrometry.

Buffers and detergents are notorious for suppression of analyte signal in electrospray and MALDI mass spectrometry and, invariably, analysts will take steps to remove these contaminants before MS analysis. However, we have found serendipitously that protein signal with MALDI MS is improved by about an order of magnitude on the addition of small amounts of Tween80. Four charged states of BSA could easily be seen at less than 125 fmol/spot and with mixture of three proteins (BSA, trypsinogen, and protein A) the molecular ions could be detected on as little as 12.5 fmol of spotted material (per protein) using an automated laser firing sequence.

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 spectro-metry 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 X100 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.