Anal. Chem. 2009, 81, 3986–3996
De Novo Sequencing of RCB-1 to -3: Peptide Biomarkers from the Castor Bean Plant Ricinus communis Simon P. B. Ovenden,*,† Sten-Åke Fredriksson,*,‡ Christina K. Bagas,† Tomas Bergstro¨m,‡ Stuart A. Thomson,† Calle Nilsson,‡ and David J. Bourne† Defence Science and Technology Organisation, 506 Lorimer Street Fishermans Bend, Victoria 3207, Australia, and Swedish Defence Research Agency, FOI CBRN Defence and Security, SE-901 82 Umea˚, Sweden
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-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. The study of the different populations of small molecules (the metabolome) from different specimens of the same genera of * To whom correspondence should be addressed. E-mail: simon.ovenden@ dsto.defence.gov.au (S.P.B.O.),
[email protected] (S.-Å.F.).
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plant, and subsequent multivariate statistical analysis (Chemometrics), can potentially provide information that will allow for determinations to be made regarding the type of cultivar/species of the plant specimen being studied.1 Advantageously, in addition to cultivar/species identification, literature evidence suggests that the geographical location of the plant may also be elucidated.2-7 These parallel analyses, and the results generated from them, have previously allowed for important conclusions to be drawn on food authenticity,8 but has also played a significant role in forensic applications such as the country of origin of illicit drugs.2-5 Importantly, for these analyses to be successful, knowing the actual content of the plant metabolome is critical. Implicitly, this means the actual chemical structure of the molecules that make up the metabolome population need to be determined. Through solving the individual constituent structures of this population, a determination can be made on which particular molecules are important as indicators of cultivar/species, and furthermore which molecules act as indicators for a specific geographical location. Therefore the structural elucidation of key chemical compounds within the metabolome is crucial. The plant Ricinus communis (also known as the castor bean plant) is the source of castor oil, historically an important oil that has found broad application in many and varied industries. It is also an invasive species that contains the Ribosome-Inactivating Protein (RIP) ricin,9 and as such poses a significant threat to humans and wildlife if it is ingested. This was demonstrated by †
Defence Science and Technology Organisation. FOI CBRN Defence and Security. (1) Periera, G. E.; Gaudillere, J. P.; Leeuwen, C. V.; Hilbert, G.; Lavialle, O.; Maucourt, M.; Deborde, C.; Moing, A.; Rolin, D. J. Agric. Food Chem. 2005, 53, 6382–6389. (2) Ehleringer, J. R.; Casale, J. F.; Lott, M. J.; Ford, V. L. Nature (London) 2000, 408, 311–312. (3) Hays, P. A.; Remaud, G. S.; Jamin, E.; Martin, Y. L. J. Forensic Sci. 2000, 45, 552–562. (4) Reddy, M. M.; Ghosh, P.; Rasool, S. N.; Sarin, R. K.; Sashidhar, R. B. J Chromatogr. A 2005, 1088, 158–168. (5) Mohana, M.; Reddy, K.; Jayshanker, G.; Suresh, V.; Sarin, R. K.; Sashidhar, R. B. J. Sep. Sci. 2005, 28, 1558–1565. (6) Brescia, M. A.; Di Martino, G.; Guillou, C.; Reniero, F.; Sacco, A.; Serra, F. Rapid Commun. Mass Spectrom. 2002, 16, 2286–2890. (7) Oliveros, C. C.; Boggia, R.; Casale, M.; Armanino, C.; Forina, M. J. Cromatogr. A 2005, 1076, 7–15. (8) Cotte, J. F.; Casabianca, H.; Lheritier, J.; Perrucchietti, C.; Sanglar, C.; Waton, H.; Grenier-Loustalot, M. F. Anal. Chim. Acta 2007, 582, 125– 136. (9) Lord, M. J.; Jolliffe, N. A.; Marsden, C. J.; Pateman, C. S.; Smith, D. C.; Spooner, R. A.; Watson, P. D.; Roberts, L. M. Toxicol. Rev. 2003, 22, 53– 64. ‡
10.1021/ac900371y CCC: $40.75 2009 American Chemical Society Published on Web 04/24/2009
the death of Georgi Markov in 1978, with ricin suspected to be the agent used for his assassination.10 Because of this, research looking at the proteome and metabolome of the seeds of R. communis has been conducted with the aim to identify both cultivar and geographic origin of specimens of R. communis from either an extract generated from a collected seed or an already available extract of a seed. During MALDI TOF and LC-MS analysis of crude aqueous acetic acid extracts from eight specimens of six cultivars of R. communis “zanzibariensis” collected from Kenya and Tanzania, “gibsonii”, “impala”, “dehradun”, “carmencita”, and “sanguineus” collected from Spain and Tanzania), the presence of three low molecular weight peptide biomarkers (designated Ricinus communis biomarkers, or RCB) was observed. Detailed analysis of the spectra identified that the specimens of cultivars of both “zanzibariensis” specimens, “gibsonii”, “impala”, “dehradun”, and both “sanguineus” specimens contained RCB-1 and RCB-2 with a molecular mass of 2066 and 1978 Da, respectively (see Figure 1a). Conversely, the extract of the “carmencita” cultivar contained relatively minor amounts of RCB-1. It did contain, however, significantly more of RCB-2, and an additional peptide biomarker, RCB-3, with a mass of 1961 Da (see Figure 1a). Reported below are the elucidated amino acid sequences of RCB-1 to -3, and the chemical manipulations performed to determine the connectivity of the two disulfide bonds present in each RCB. Also discussed is the possible functional role of RCB-1 to -3 in the seeds as potential antimicrobial chemoprotective agents. EXPERIMENTAL SECTION General Experimental Information. Methanol (MeOH), acetonitrile (MeCN), H2O, trifluoroacetic acid (TFA), and formic acid were HPLC grade and obtained from Merck (Melbourne, Australia). Dithiolthreitol (DTT), iodoacetamide (IAM), ammonium bicarbonate, tris-(2-carboxyethyl)phosphine (TCEP), and N-ethylmaleimide (NEM) were AR grade and obtained from Sigma-Aldrich (Sydney, Australia). Bulk C18 for column chromatography was obtained from Phenomenex (Sydney, Australia). Empty solid phase extraction (SPE) cartridges with a capacity of 1 mL were obtained from Grace Davison (Melbourne, Australia). Molecular Weight Cut Off (MWCO) filters (Amicon ultra 15 mL) used were for a molecular weight cutoff of 30 kDa and obtained from Millipore (Australia). The MWCO filters were spun using a Sigma 3-15 Laboratory Centrifuge from Quantum Scientific (Brisbane, Australia). Concentration of samples was performed using a Thermo Savant SPD 111V Speed Vac centrifugal concentrator (Waltham, U.S.A.). The proteases trypsin and chymotrypsin were sequencing grade and obtained from G-bioscience (Sydney, Australia). ESI LC-MS Analysis. LC-MS and LC-MSn studies were performed on either of following two instruments. Instrument one was an Agilent LC/MSD Trap XCT mass spectrometer connected to an Agilent 1100 series LC system (Melbourne, Australia) comprising an in-line degasser, binary pump, autoinjector, column heater, and diode array detector, equipped with (10) Darby, S. M.; Miller, M. L.; Allen, R. O. J. Forensic Sci. 2001, 46, 1033– 1042.
Agilent ChemStation LC for 3D software (Rev.A.09.03). Samples were eluted through a Phenomenex Gemini 5 µm 50 × 2.0 mm C18 HPLC column, using gradient elutions of either 8 or 30 min, from H2O (+0.05% formic acid) to 70% MeOH in H2O (+0.05% formic acid). The mass spectrometer was operated in “smart mode”, with target masses set at either m/z 600, 900, and 1500. Nitrogen was used as nebulizer and desolvation gas with argon used as the collision gas. Instrument two was a Micromass quadrupole time-of-flight (Q-TOF) Ultima hybrid mass spectrometer with a nanospray ion source and a CapLC chromatographic system (Waters, Manchester, U.K.). Samples were separated using a Pepmap C18 100 mm × 75 µm nanoflow LC column (Dionex, Sunnyvale, U.S.A.) with gradient elution from 5 to 60% MeCN in H2O (+0.2% formic acid). A Picotip nanospray emitter (New Objective, Woburn, U.S.A.) was operated at about 1.8 kV. Nitrogen was used as nebulizer and desolvation gas. MS/MS product ion spectra were recorded using collision energy between 15 and 35 V with argon as the collision gas. Fourier Transform Mass Spectrometry. High resolution Fourier Transform Ion Cyclotron Resonance Mass Spectrometry (FTICR-MS) data was collected on a Bruker Apex Ultra FTMS, equipped with a 9.4 T magnet, ESI, and MALDI sources. Peptides dissolved in MeOH (+1% formic acid) were directly infused into the mass spectrometer via a syringe pump for ESI analysis. Sustained Off Resonance Induced (SORI) dissociation was carried out by in-cell isolation of the precursor ion followed by SORI using a pulse time of 100 ms and RF energy of about 40 dB at a frequency offset by 500 Hz from the precursor ion cyclotron resonance frequency. Ultra High purity argon was used as collision gas. MS/MS calibration was applied externally using a cluster from methanolic NaOH/trifluoroacetate solution as a precursor ion and carrying out SORI dissociation under the same conditions. A matt steel MALDI target was prepared by co-crystallization of crude castor bean extracts and 4-hydroxy-R-cyano-cinnamic acid (HCCA) matrix. Spectra obtained from these spots were calibrated against a standard peptide mixture of angiotensin II, substance P, bombesin, ACTH clip 1-17, and somatostatin 28 (Bruker, Bremen, Germany). MALDI MS. MALDI data was generated on a Bruker AuTOFlex II MALDI TOF/TOF instrument (Leipzig, Germany) and flexControl software for acquisition and flexAnalysis for data manipulation. MALDI target preparation was identical to that described for high resolution FTICR-MS. Extraction of R. communis Beans. Whole R. communis beans (∼ 1 g from each specimen) were ground with a mortar and pestle, and treated with acetone (∼20 mL) stirring overnight at 4 °C to remove the castor oil. The acetone was removed via filtration, with the residual mash treated with 20 mL of 2% acetic acid by vigorous shaking and allowed to stand for 1 h. The mash was again filtered to yield an enriched protein extract of each specimen of R. communis in 2% acetic acid. The extract was centrifuged at 3000 rpm for 2 h through a
