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, carmen-cita, and sanguineus collected from Spain and Tanzania), three peptide biomarkers (designated Ricinus communis biomarkers, or RCB) were identified in both the MALDI and electrospray LGMS 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-MSn 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.