Anal. Chem. 2007, 79, 6271-6278
Solvent-Assisted Trypsin Digestion of Ricin for Forensic Identification by LC-ESI MS/MS Anders O 2 stin,* Tomas Bergstro 1 m, Sten-A ° ke Fredriksson, and Calle Nilsson
FOI, Swedish Defence Research Agency, CBRN Defence and Security, Umea˚, SE-901 82, Sweden
The castor bean plant (Ricinus communis) is used in large quantities for oil production and is also a common ornamental garden plant. However, the beans contain 1-3% of the highly toxic protein ricin, a type II ribosomeinactivating protein that is covered by the Chemical Weapons Convention, and there have been a number of reports concerning the use, or alleged use, of the toxin in terrorist and criminal activities. In the study reported here, we investigated the potential utility of organic solvent-assisted trypsin digestion of crude extracts containing the closely related toxins ricin or abrin to prepare samples for peptide analysis by liquid chromatography combined with electrospray ionization quadrupole timeof-flight tandem mass spectrometry. Diagnostic tryptic fragments of the toxins were detected and unambiguously identified by this procedure. The sample preparation protocol substantially reduces the sample preparation time, from overnight to an hour, and thus greatly reduces the total time required for analyses, to less than 2 h. Furthermore, the reported procedure leaves the disulfide bonds in the protein intact. This is highly relevant in the context of the Chemical Weapons Convention, since the disulfide bond connecting the two chains of ricin indicates the presence of an intact toxin and provides additional forensic evidence for the analytical results. The beans of the castor bean plant (Ricinus communis) contain the highly toxic protein ricin at concentrations of 1-3%. Ricin is a type II ribosome-inactivating protein (RIP II) with a molecular mass of ∼64 000 Da. At a molecular level, RIP II proteins consist of two polypeptide chains, designated the A- and B-chains. The B-chain can bind to the surface of cells where it comes into contact with carbohydrate-binding sites and thereby facilitates endocytosic uptake of the protein. Once inside a cell, the interchain disulfide bond is reduced and the released A-chain catalytically inactivates the ribosomes.1-3 Poisoning leads initially to flu-like symptoms, and death may ensue within a few days following exposure. There are no known effective antidotes for treating ricin poisoning.4-6 * To whom correspondence should be addressed. E-mail: anders.ostin@ foi.se. (1) Hartley, M. R.; Lord, J. M. Biochim. Biophys. Acta: Proteins Proteomics 2004, 1701, 1-14. (2) Lord, J. M.; Roberts, L. M.; Robertus, J. D. FASEB J. 1994, 8, 201-208. (3) Stirpe, F. Toxicon 2004, 44, 371-383. (4) Bigalke, H.; Rummel, A. Toxicology 2005, 214, 210-220. (5) Bradberry, S. M.; Dickers, K. J.; Rice, P.; Griffiths, G. D.; Vale, J. A. Toxicol Rev. 2003, 22, 65-70. 10.1021/ac0701740 CCC: $37.00 Published on Web 07/11/2007
© 2007 American Chemical Society
RIP II toxins are also found in other plants. Examples of such related toxins (and their sources) include abrin (Abrus precatorius),7,8 volkensin (Adenia volkensii),9 and viscumin (Viscum album).10,11 The Chemical Weapons Convention (CWC)12 came into force after being signed by 87 State Parties in 1997 and applies in principle to “any chemical which through its chemical action on life processes causes death, temporary incapacitation or permanent harm to humans and animals”. Its aims are to eliminate chemical weapons, by banning their production or use, and enforcing the destruction of any such weapons that have been produced. In addition to clauses regarding these aims, it provides schedules of known chemical warfare agents, their precursors, and harmful industrial chemicals that are subject to inspection and control measures according to specified procedures. Ricin is the only protein listed in Schedule 1 (chemical weapons) of the CWC. Any extraction of ricin from plant material should thus be declared to the Organization for the Prohibition of Chemical Weapons (OPCW) according to the CWC as long as the A- and B-chains are connected with an intact disulfide bond.12,13 Prior to the convention, there were reports of trials with weaponized ricin in several countries, but no extensive ricin weapon production seems to have occurred, probably because of the unsuitable dispersion properties of ricin.14,15 However, during the past decade, there have been a number of cases in which ricin has been used in smallscale incidents or criminal activities, so there is a clear need for accurate methods that can unambiguously identify the toxin.15,16 (6) Dickers, K. J.; Bradberry, S. M.; Rice, P.; Griffiths, G. D.; Vale, J. A. Toxicol Rev. 2003, 22, 137-142. (7) Chen, Y. L.; Chow, L. P.; Tsugita, A.; Lin, J. Y. FEBS Lett. 1992, 309, 115118. (8) Funatsu, G.; Taguchi, Y.; Kamenosono, M.; Yanaka, M. Agric. Biol. Chem. 1988, 52, 1095-1097. (9) Stirpe, F.; Barbieri, L.; Abbondanza, A.; Falasca, A. I.; Brown, A. N.; Sandvig, K.; Olsnes, S.; Pihl, A. J. Biol. Chem. 1985, 260, 14589-14595. (10) Soler, M. H.; Stoeva, S.; Schwamborn, C.; Wilhelm, S.; Stiefel, T.; Voelter, W. FEBS Lett. 1996, 399, 153-157. (11) Soler, M. H.; Stoeva, S.; Voelter, W. Biochem. Biophys. Res. Commun. 1998, 246, 596-601. (12) Convention on the prohibition of the development, production, stockpiling and use of chemical weapons and their destruction. www.opcw.org. Entry into force, April 29, 1997. (13) OPCW; the Technical Secreteriat for the Organisation for the Prohibition of Chemical Weapons, Scientific Advisory Board, 1999; SAB-II/-1. (14) Craig, H. L.; Alderks, O. H.; Corwin, A. H.; Dieke, S. H.; Karel, C. L.; U.S. Patent 3,060,165, 1952. (15) Lundberg, S.; Melin, L.; Nilsson, C.; Schoenberg, P. v. NBC-Defence, Ed.; FOI Swedish Defence Research Agency, 2004. (16) Olsnes, S. Toxicon 2004, 44, 361-370.
Analytical Chemistry, Vol. 79, No. 16, August 15, 2007 6271
Current methods for unambiguous identification of ricin include molecular weight determinations by MALDI-MS to provide proof of intact disulfide bridges between the A- and B-chains, and trypsin digestion of the sample, a procedure that normally involves denaturation with a chaotropic agent, reduction of disulfide bridges, and alkylation of the resulting thiols, followed by overnight trypsin digestion. On the following day the sample is analyzed by LC-MS peptide mapping and MS/MS of selected specific peptides.17 In addition, MALDI-MS analysis and in vivo toxicity testing may be used. As an indirect indicator of the presence of ricin, the alkaloid ricinine has been proposed as a marker in crude plant extracts, urine from potentially exposed persons, or both.18,19 In the study reported here, the possibility of reducing the time required for unambiguously identifying ricin using organic solventassisted trypsin digestion20-22 was evaluated. The use of methanol and isopropanol for this purpose was investigated, and conditions were optimized for the proteolysis of native ricin by trypsin. The optimized protocol was subsequently tested on crude extracts of castor beans (R. communis), and of A. precatorius seeds containing abrin, a related RIP II-toxin. EXPERIMENTAL SECTION Safety Considerations. Since ricin is listed in Schedule 1 of the Chemical Weapons Convention, states that have ratified the CWC are required to declare the production (extraction) and possession of ricin (also in small-scale laboratory) to the OPCW.(12) The proteins in this study, ricin and abrin, are toxins that inhibit protein synthesis in cells. Due to their high toxicity, it is recommended that all work performed with intact toxins be done in a specially designated laboratory. All contact with the active toxins should be avoided, and it is recommended to keep the toxins in solution to avoid dust formation. In this study, laboratory material, including single-use material, was decontaminated by submersion in 2 M NaOH. Materials and Chemicals. Ricin was isolated from R. communis, var. zanzibariensis castor beans supplied by Rara Va¨xter (Stockholm, Sweden) and prepared in-house according to Fredriksson et al.17 A. precatorius seeds were bought in an open market in Egypt. Crude extracts of ricin and abrin were prepared from 0.5 g of the castor beans and A. precatorius seeds, respectively, as follows. The seeds were homogenized in a mortar and lipids were removed using ethyl acetate. The toxins were then extracted using 4 mL of water at pH 4 (acidified with acetic acid). The extracts were centrifuged at 14 000 rpm for 10 min, and the resulting supernatants were used as crude toxin extracts. Molecular weight cutoff spin filter (MWCO filter) with a 10 000 NMWL cutoff (Amicon ultra free-MC centrifugal filter, Biomax10, from Millipore, Bedford, MA) was used for protein concentration (see below). (17) Fredriksson, S. A.; Hulst, A. G.; Artursson, E.; de Jong, A. L.; Nilsson, C.; van Baar, B. L. M. Anal. Chem. 2005, 77, 1545-1555. (18) Darby, S. M.; Miller, M. L.; Allen, R. O. J. Forensic Sci. 2001, 46, 10331042. (19) Johnson, R. C.; Lemire, S. W.; Woolfitt, A. R.; Ospina, M.; Preston, K. P.; Olson, C. T.; Barr, J. R. J. Anal. Toxicol. 2005, 29, 149-155. (20) Russell, W. K.; Park, Z. Y.; Russell, D. H. Anal. Chem. 2001, 73, 26822685. (21) Soskic, V.; Godovac-Zimmermann, J. Proteomics 2001, 1, 1364-1367. (22) Welinder, K. G. Anal. Biochem. 1988, 174, 54-64.
6272
Analytical Chemistry, Vol. 79, No. 16, August 15, 2007
Protein Amino Acid Sequence Information. Sequence information was obtained from the Swissprot database on ricin, R. communis agglutinin, and abrin-a, -b, -c, and -d: entries P02879, P06750, P11140, Q06077, P28590, and Q06076, respectively23 Computer Software. MODDE ver. 6.0 (Umetrics, Umeå, Sweden) was used to generate experimental designs and evaluate the acquired data. Solvent-Assisted Trypsin Digestion Optimization. In order to screen for optimum conditions for trypsin digestion of native ricin, the importance of digestion time, concentration of organic solvent, trypsin content, and temperature was evaluated using a reduced central composite face (CCF) design (Supporting Information, Table 1). CCF designs consist of a full or fractional design and star points placed on the faces of the sides.24,25 Limits for the model were as follows: organic solvent (methanol and 2-propanol), 20-80%; amount of trypsin, 0.1-10 µg/sample; temperature, 2555 °C; and digestion time, 15-65 min. Each of the samples contained 12.5 µg of toxin in 100 µL of 50 mM NH4HCO3. Digestion was terminated by acidifying the sample with formic acid. The samples were concentrated to dryness and dissolved in 50 µL of HPLC mobile phase before LC-MS analysis (see below). In MODDE, there are two alternatives for creating a model: multiple linear regression or the partial least-squares (PLS) method.24,25 The latter was used here since there are several correlated responses in the data set. The response data consisted of the chromatographic peak areas of the most intense ricin trypsin digest peptides, marked by an (f) in Tables 1 and 2. The TA2021 (doubly and triply charged) peptide responses were also included. The data, in total 16 relative responses, were logtransformed and nonsignificant coefficients were removed. Enzymatic Digestion of Crude Extracts Containing Ricin and Abrin and Sample Preparation for MS. Portions (100 µL) of the crude extracts were prepared using a 0.5-mL Zeba gel filtration centrifugal cartridge with size exclusion limit of 7000 Da (Pierce, Rockford, IL) (conditioned with aqueous digestion buffer). Trypsin was added to give an enzyme-to-substrate ratio of ∼1:2, and methanol was added to a final concentration of 50% to assist the digestion. The substrate concentration was calculated by assuming that ricin (or abrin, as appropriate) accounted for 1% of the total weight of the plant material used. The digestion was performed at 25 °C for 65 min and was then terminated by the addition of 5 µL of formic acid. Prior to LC-MS analysis, the sample was concentrated to dryness and diluted to 50 µL with 95:5 water/acetonitrile containing 0.2% formic acid. CE-UV Analysis. For capillary electrophoresis (CE) analysis of different reaction mixtures, the system used comprised an Agilent capillary electrophoresis instrument (Palo Alto, CA) equipped with a UV diode-array detector, ChemStation software for data collection, and a fused-silica capillary with a total length of 34 cm, effective length 26 cm, 50-µm i.d., and 375-µm o.d. The buffer was a 50 mM phosphate buffer, pH 2.5. Settings were as follows: temperature, 15 °C; injection duration, 8 s; injection pressure, 50 mbar; separation voltage, 17 kV; ramp time, 1 min. (23) Swissprot; www.expasy.org/sprot/, Swiss Institute of Bioinformatics: Geneva, Switzerland. (24) Eriksson, T.; Johansson, E.; Kettaneh-Wold, N.; Wols, S. Design of Experiments. Principles and Applications; Umetrics AB: Umea˚, 2000. (25) Eriksson, T.; Johansson, E.; Kettaneh-Wold, N.; Wols, S. Multi- and megavariate data analysis. Principles and applications.; Umetrics AB: Umeå, 2001.
Table 1. Trypsin Digest Peptides from Chain A of Purified Ricin tryptic fragmenta
precursor ionb
CEc
TA1 TA2 (Gx)
504.31+ 1225.93+
28 33
IFPR QYPIINFTTAGATVQSYTNFIR
TA3 TA4 TA5
{344.2} {231.1} 832.41+
43
AVR GR LTTGADVR
TA5 TA6 TA7 TA8
416.72+ 537.82+ f 448.82+ f 1069.63+
28 28 26 26
HEIPVLPNR VGLPINGR FILVELSNHAELSVTLALDVTNAYVVGYR
TA9
827.64+ f
28
AGNSAYFFHPDNQEDAEAITHLFTDVQNR
TA9
1103.23+
48
TA10 TA10-11 TA11 TA12
655.82+ 769.13+ f 507.32+ 1147.63+ f
28 28 28 28
YTFAFGGNYDR YTFAFGGNYDRLEQLAGNLR LEQLAGNLR ENIELGNGPLEEAISALYYYSTGGTQLPTLAR
TA13 TA14 TA15 TA16 TA17 TA18 TA19
791.42+ 586.82+ {275.2} {287.2} 452.21+ {174.1} 864.92+ f
28 28
SFIICIQMISEAAR FQYIEGEMR TR IR YNR R SAPDPSVITLENSWGR
TA20 TA20
1130.12+ f 753.73+ f
38 28
TA21 TA22 TA23
{174.1} {404.2} 1106.62+
28 28
TA23
738.13+
28
TA24-ss-TB1
759.03+
28
TA24-ss-TB1
1138.02+
33
28 28
amino acid sequenced
observed sequence ionse y′′1-y′′3, b2, a2, FP y′′1-y′′11, b5, TA22+, [TA2+HexNAc+2H] 2+, HexNAc, (HexNAcHex1)-(HexNAcHex3) y′′1-y′′2, z1-z2, TGAD, DV, [M+H-H2O]1+, [M+H-CO2] 1+ y′′1-y′′2, b2/DV, z1-z2, a2/AD, a4/TGAD, GAD y′′1, y′′3-y′′8, b1-b3, a1-a3, z3-z7, PV y′′1-y′′7, b2, z1-z5, PL/LP, NQ, GLP, PIN y′′1-y′′14, b182+-b212+, b232+-b282+, [M+3H-H2O]3+, y′′1, y′′4- y′′12, y′′92+-y′′132+, y′′202+- y′′212+, y′′223+-y′′283+, b3-b5, NS, PD-H20, FH, GNSA, GNSA-NH3, QED-NH3, FFH y′′1, y′′3-y′′15, y′′202+-y′′242+, b3-b6, b9, b12, b14-b15 y′′1, y′′4-y′′10, b4, i1, i7/i9 y′′1, y′′4-y′′7, y′′122+-y′′192+, b2-b4, a1-a2 y′′1- y′′2, y′′3-y′′8, b2, z7-z8, AGN-CO y′′1-y′′14, b4-b5, b14, b17, b172+-b212+, b242+- b272+, [M+3H-H2O]3+ y′′1, y′′3-y′′12, b2-b6, a2 y′′1-y′′2, y′′4-y′′8, z1, z4-z8, c1 y′′1, a1 y′′2-y′′14, y′′122+, y′′142+, b3-b6, PD/ DP, PDPSVI y′′1-y′′9, y′′11-y′′16, b4-b7, b10, b12-b15 y′′1-y′′10, y′′62+-y′′102+, b2-b6, i10/20, i21,PI, AF/FA, IQ/QL/LQ, PQN
LSTAIQESNQGAFASPIQLQR R NGSK FSVYDVSILIPIIALMVYR
y′′1-y′′7, y′′9-y′′15, b3-b10, VYDVS-H20/SVYDV-H20, SILIPIIA-CO, VSILIPIA-CO, VSILIPIIAL-CO, DVSILIPIIAL-CO y′′1-y′′11, SI-H20, IP/PI, YDVS-H20/SVYD-H20, VSILI-CO, VYDVS-H20/SVYDV-H20, VSILIPIIA-H20
Disulfide-Linked A-Chain TA24 and B-Chain TB1 ADVCNDPEPIVR [TB1] CAPPPSSQF [TA24]
[TB1+TA24b2], [TB1+TA24b3-TA24b8], TB1; y′′1, y′′4-y′′8, TB24; y′′1-y′′3, y′′5-y′′7, SS, PP, SQ, PPPS, APPPSSQ [TB1-ss-TA24b2], [TB1b7-ss-TA24], [TB1-ss-TA24b8]2+, [TB1y′′10-ss-TA24]2+, [TB1-ss-TA24b8]2+, TB1;y′′6, TA24; y′′6-y′′7
a Tryptic peptides of ricin numbered from the amino terminal. b In boldface type; peptides differentiating ricin from RCA.35-37 In brackets, ions not observed in LC-MS given as theoretical mass. c Optimized collision energy for MS/MS analysis. d Theoretical amino acid sequence for ricinD. In boldface type, disulfide-linked cysteines. e Observed sequence ions from MS/MS analysis with annotation of sequence ions according to Roepstorff and Fohlman.38 f Used as a response in the design of experiments. Abbreviations: TchainX, peptide with chain notation; TchainX-Y, chain with missed cleavage peptide; TchainX-ss-TchainX, disulfide-linked peptide; Gx, glycopeptide.
Eluting substances were detected by monitoring absorption at 195 nm. The capillary was preconditioned by flushing with 0.5 M HCl for 1.5 min, followed by 0.5 M NaOH for 1.5 min, MilliQ water for 4 min, and, finally, separation buffer for 3 min. LC-ESI-MS/MS Snalysis. The HPLC system used for this analysis was a CapLC instrument (Waters, Milford, MA). Samples (1 µL) were injected by the “µL pick up” routine and separated on a 0.3 × 150 mm PepMap300 capillary column, particle size 5 µm, 300-Å pore size (LC Packings, Amsterdam, The Netherlands). Samples were eluted using a two-step, 7 µL/min gradient starting with a linear increase from 5 to 10% acetonitrile over 4 min, followed by an increase from 10 to 80% acetonitrile over 42 min, with 0.2% formic acid and 0.02% triflouroacetic acid throughout
as additives for ion suppression. The column was connected to a Micromass Ultima QTOF hybrid quadrupole time-of-flight mass spectrometer (Waters) equipped with a standard electrospray ion source. The source was operated at 3.1 kV with a cone voltage of 35 V. Nitrogen was used as nebulizer and desolvation gas and argon as collision gas. In MS and MS/MS analyses, the collision energies were set to 10 and 28-48 V, respectively. The scan range was m/z 300-1800 for peptide mass mapping and m/z 100-1800 for product ion spectrum acquisition. MassLynx software (Waters) was used for instrument control and data processing. These scan conditions were then combined in data-dependent analysis (DDA). In DDA mode, the QTOF continuously makes survey scans, and when a peptide with a preprogrammed m/z value is detected, a Analytical Chemistry, Vol. 79, No. 16, August 15, 2007
6273
Table 2. Trypsin Digest Peptides from Chain B of Purified Ricin CEc
amino acid sequenced
tryptic fragmenta TB1 TB2 TB3-ss-TB5
444.31+ 800.73+ f
28 28
TB3-ss-TB5
600.84+
28
TB4 TB6 TB6-7 TB7 TB8 TB9 TB10
{346.2} 695.82+ 516.33+ {174.3} 618.31+ {404.2} 983.13+
TB11 (Gx)
997.83+
TB12
{6934.3}
TB13 TB13
1116.92+ 744.43+
48 28
TB14-ss-TB16
804.03+
33
TB14-ss-TB16
603.34+ f
33
TB15 TB17 TB17-18 TB18 TB18 TB19 TB19
575.31+ 611.31+ 818.83+ 931.52+ f 621.33+ f 717.41+ 359.22+
28 26 28 28 28 33 26
ETVVK WMFK WMFKNDGTILNLYSGLVLDVR NDGTILNLLYSGLVLDVR
TB20 TB20
1139.12+ f 759.73+ f
28 28
QIILYPLHGDPNQIWLPLF
precursor ionb
IVGR NGLCVDVR [TB3] FHNGNAIQLWPCK [TB5]
33
DGR SNTDANQLWTLK SNTDANQLWTLKR R DNTIR SNGK CLTTYGYSPGVYVMIYDCNTAATDATR
28
WQIWDNGTIINPR
28 28 28
SSLVLAATSGNSGTTLTVQTNIYAVSQGWLPTNNTQPFVTTIVGLYGLCLQANSGQVWIEDCSSEK AEQQWALYADGSIRPQQNR DNCLTSDSNIR [TB14] ILSCGPASSGQR [TB16]
ASDPSLK
observed sequence ione disulfide bond, see Table 1 y′′1-y′′3, b2-b3, a2 [M+3H-H2O]3+, [TB3-ss-TB5y′′3-TB5y′′4]1+, [TB3-ss-TB5b12]3+, [TB5-ss-TB3y′′5]2+, TB3; y′′1-y′′2, TB5; b2-b3, b6-b10, i10 [TB3-ss-TB5y′′2-y′′3]1+, TB3; no fragments TB5; b6-b8, i10, IQ/QL-H20, IQ/QL-C0, NGN-H20, HNGA, NGNAIQ-CO y′′1-y′′10, b2-b3, z5, [M+2H-H2O]2+ y′′1- y′′8, b2, z2-z8, i3, i9, TD, NQ y′′1- y′′2, y′′4, b2-b3, z4, TI, y′′1- y′′9, i5/i16, PG, PGV, PGV, PGVY, PGVYV, PGVYVM, PGVYVMI, PGVYVMIY, [M+3H-H2O]3+, ([y′′-b]2+)g y′′2- y′′5, b2-b5, INP, T11, T112+, [TB11 HexNAc+2H]2+, [TB11+ 2HexNAc+ 2H]2+, [TB11+2HexNAc Hex+2H]2+[TB11+HexNAc+4Hex + 2H]2+, HexNAc, (HexNAc-Hex1)-(HexNAc-Hex3)
no fragmentation b2-b6, y′′1, y′′5, y′′92+-y′′172+, DG, QQ, EQ, EQQ-NH3, QQ-NH3, QQWA-NH3 [TB14-ss-TB16y′′9-y′′11]2+, [TB16-ss-TB14y′′9]2+, TB14; y′′1-y′′8, TB16; y′′1-y′′8, b2-b3, a2 TB14; y′′1-y′′2, y′′4-y′′7, b2, NCL, TB16; y′′1-y′′11, b2, a2 y′′1-y′′4, b2-b3 y′′1-y′′3, b2, a2 y′′2-y′′5, y′′7-y′′11, b2, b112+-b132+, ILN/LNL y′′1-y′′13, b2-b10 y′′1-y′′5, y′′7-y′′9, b2, b4, b7-b8, i9, LN/NL, SGL y′′2-y′′5, b3, b5, PS, PSL-H2O y′′1-y′′3, b3, z1, PS/SD-H2O, PS-CO, PS- H2O, SL-CO, DP-H2O, DP, PSL-H2O, DPS-H2O/SDP-H2O, PSL, y′′2-y′′5, y′′14, b14-b15, b142+-b182+ y′′1-y′′4, b2-b4, b112+-b162+, i15
a Tryptic peptides of ricin numbered from the amino terminal. b In boldface type; peptides differentiating ricin from RCA.35-37 In brackets, ions not observed in LC-MS given as theoretical mass. c Optimized collision energy for MS/MS analysis. d Theoretical amino acid sequence for ricin-D. In boldface type, disulfide-linked cysteines. e Observed sequence ions from MS/MS analysis with annotation of sequence ions according to Roepstorff and Fohlman.38 f Used as a response in the design of experiments. g Extensive fragmentation of T10 with intact disulfide bridge giving a combined doubly charged y′′1+ and b1+ fragment ([y′′ - b]2+) most abundant; [y′′12 + b7]2+, [y′′12 + b8]2+, [y′′13 + b8]2+, [y′′14 + b7]2+, [y′′14 + b8]2+, [y′′12 + b10]2+, [y′′13 + b11]2+, [y′′12 + b12]2+, [y′′16 + b8]2+, [y′′11 + b13]2+, [y′′12 + b7]2+, [y′′12 + b14]2+, [y′′24 + b2]2+/[ y′′23 + b3]2+/y′′262+. Abbreviations: TchainX, peptide with chain notation; TchainX-Y, chain with missed cleavage peptide; TchainX-ss-TchainX, disulfide-linked peptide; Gx, glycopeptide.
product ion spectrum is automatically acquired at elevated collision energy (Tables 1 and 2). Ricin- and abrin-specific tryptic peptides were specified in an include list (Supporting Information Table 3), which contained information on m/z values and collision energies. DDA was performed on crude extracts of castor beans and seeds from A. precatorius. RESULTS AND DISCUSSION Solvent-Assisted Trypsin Digestion Optimization. One way in which the time required to prepare native, untreated proteincontaining samples for analysis can be substantially shortened is to apply organic solvent-assisted trypsin digestion, using solvents compatible with HPLC-ESI MS.20-22 The concentration of organic solvent when using this approach should be optimized for each type of protein. In order to identify suitable conditions for trypsin digestion of native ricin, we used a reduced CCF experimental design for optimization and a purified ricin standard. The optimized conditions were then applied to prepare crude toxin samples. 6274 Analytical Chemistry, Vol. 79, No. 16, August 15, 2007
The model obtained for methanol-assisted digestion contains four PLS components with an R2 value of 0.92 and Q2 value of 0.80. The responses for individual peptides show R2 values ranging from 0.82 to 0.96 and Q2 values ranging from 0.69 to 0.87. The difference between the R2 and Q2 values was less than 0.2 for all peptides. The model is composed of the following coefficients in descending order of influence on the amount of peptides formed: trypsin, MeOH × MeOH, trypsin × trypsin, temperature, time × MeOH, MeOH, time × trypsin, and time. The first four coefficients influence the model most strongly.24,25 These findings collectively provide strong indications that our model is acceptable. The model predicts that a good enzyme-to-substrate ratio is 1:2, that the methanol content should be 50%, and that the digestion should be performed at 25 °C. The results from the screening of conditions for the solvent-assisted trypsin digestion obtained in this study demonstrated that using methanol considerably reduced the time required for adequate digestion. A similar evaluation was
performed with 2-propanol, for which the optimal enzyme/ substrate ratio proved to 2:1 in the presence of 80% 2-propanol. Under these conditions, the digestion of ricin was similarly rapid (data not shown). However, due to the advantages of using methanol in preparation of samples prior to HPLC, methanol was used in all further investigations. Trypsin peptides originating from autodigestion are formed under the optimized conditions and were found in an ∼1:1 ratio to the ricin peptides. However, they did not cause any serious interference in the verification procedure. Final conditions employed for solvent-assisted trypsin digestion were as follows: temperature, 25 °C; solvent, methanol/water (50: 50) containing 50 mM NH4HCO3; and an enzyme-to-substrate ratio of 1:2. One hour is sufficient time for quantitative digestion. Capillary electrophoresis analysis of trypsin digestion mixtures demonstrated that native purified ricin is highly resistant to trypsin digestion in an aqueous buffer, the amounts of ricin present being unaffected after 1 h, even though partial loss of trypsin due to autolysis was observed (Figure 1A and B). The addition of methanol strongly promoted the enzymatic digestion of ricin; tryptic peptides of ricin were observed within minutes, and within 1 h most of both the trypsin and ricin had been digested (Figure 1C). The obtained ricin tryptic peptides were identified by LCESI MS (Figure 2). In control experiments in which trypsin was omitted from the organic solvent-assisted trypsinolysis mixture, ricin also apparently disappeared, concomitantly with the formation of a white precipitate in the sample vial. However, application of trypsin to the precipitate generated tryptic ricin peptides and resulted in disappearance of the precipitate. From these observations, it was concluded that the addition of organic solvent caused the unfolding, denaturation, and subsequent precipitation of ricin and that trypsin can digest the denatured, and precipitated, ricin. The occurrence of tryptic peptides was detected within minutes, and intact ricin was totally depleted within 1 h. An on-line trypsin digestion reactor with bound trypsin, as described by Slysz and Schriemer, would of course be an attractive alternative.26,27 However, as discussed above, the conditions used for solvent-assisted digestion caused precipitation of the toxin, in accordance with our experience of severe carryover when attempting to use an on-line system for ricin analysis (data not shown), which thus is not a viable alternative unless the trypsin reactor is to be used only once. Peptide Mapping of Ricin. Peptide mapping analyses were performed using samples of purified ricin digested using the optimized methanolic conditions. Potential ricin tryptic peptides were identified from the amino acid sequence of ricin in the SwissProt database. The extracted ion chromatograms of the individual peptides are shown in Figure 2. A low concentration of trifluoroacetic acid (TFA) was used in the mobile phase in addition to formic acid, since the strong ion-pairing ability of TFA was required for acceptable chromatographic behavior of some of the larger tryptic peptides (e.g., A-chain TA8 and B-chain TB10). The TFA concentration was kept low to avoid suppressing ionization. For each of the potential ricin peptides, the product ion spectrum was acquired using optimized collision energies between 26 and 48 V. (26) Slysz, G. W.; Schriemer, D. C. Anal. Chem. 2005, 77, 1572-1579. (27) Slysz, G. W.; Schriemer, D. C. Rapid Commun. Mass Spectrom. 2003, 17, 1044-1050.
Figure 1. Electropherograms obtained from CZE separation of digestion mixtures. (A) Ricin and trypsin mixed at a 2:1 ratio in 40 mM ammonium bicarbonate injected immediately after mixing. (B) The same mixture as in (A) injected after 60-min incubation at 25 °C. (C) The same reaction mixture as in (A) and (B), following incubation in 50% methanol at 25 °C for 1 h. The results of LC-ESI-MS analysis of C are shown in Figure 2. Abbreviations: IS, internal standard (histamine); *, trypsin; and R, ricin.
The product ion spectra were evaluated and sequence ions are listed in Tables 1 and 2. The ricin used in this study was extracted from seeds of R. communis, variety zanzibariensis, which is known to contain only ricin-D.28 Other varieties also contain ricin-E, which differs from ricin-D in the C-terminal of chain B. It has been proposed that ricin-E is a gene recombination product of ricin-D and R. communis agglutinin (RCA).29-31 RCA occurs together with ricin in the castor bean, and both are biosynthesized as a single polypeptide chain. The proteins are post-translationally processed into an A- and a B-chain, which are linked by a disulfide bridge. The sequence homology between ricin-D and RCA is 93% for the A-chain and 84% for the B-chain, and due to the strong homology (28) Lin, T. T.; Li, S. L. Eur. J. Biochem. 1980, 105, 453-459. (29) Araki, T.; Funatsu, G. Biochim. Biophys. Acta 1987, 911, 191-200. (30) Araki, T.; Funatsu, G. FEBS Lett. 1985, 191, 121-124. (31) Araki, T.; Yoshioka, Y.; Funatsu, G. Biochim. Biophys. Acta 1986, 872, 277285.
Analytical Chemistry, Vol. 79, No. 16, August 15, 2007
6275
Figure 2. Ion chromatograms of a peptide mapping of a purified ricin standard after methanol-assisted trypsin digestion. Abbreviations: TchainX, tryptic fragment with ricin chain notation; TchainX-Y, chain with missed trypsin cleavage; TchainX-ss- TchainX, disulfide-linked peptide.
between the two proteins, care must be applied to avoid false positive identification of RCA as ricin. Verification of Ricin-A-Chain Tryptic Peptides. Using the solventassisted trypsinolysis protocol, 17 out of 24 possible tryptic fragments from the A-chain were verified by MS/MS (Table 1), collectively corresponding to 96% of the total sequence. Tryptic fragments with sequence differences from RCA are highlighted in Tables 1 and 2. The remaining peptides have sequences identical to that of RCA. A-chain TA24 is linked by a disulfide bond to B-chain TB1. Two peptides, TA2 and TA22, contain the Nglycosylation consensus sequence N-X-S/T. TA2 was detected as a glycopeptide with [M + 3H]3+ at m/z 1225.9. TA22 was not detected either with or without glycosylation, in accordance with previous results obtained using the reduction and alkylation technique.17 A number of short peptides were also not detected, most likely due to their high polarity and consequently weak chromatographic retention. They each contain only 1-3 amino acids and provide limited sequence information. The large peptides TA8 and TA23 were found when solventassisted trypsin digestion was used. They were not reportedly detected when a standard digestion protocol was used together with a centrifugal MWCO-filter.17 The A-chain was analyzed using the Kyte-Dolittle hydropathic index,32 and it was shown that TA8, TA13, and TA23 are the most hydrophobic parts of the protein (data not shown). When the solvent-assisted digestion procedure was applied to a sample that was concentrated on a MWCO filter, TA8 and TA23 were lost, despite washing the filter with the organic solvent, indicating that this may be a MWCO filter-related problem. The detection of TA8 and TA23 are the main reasons for the improved sequence coverage observed in this study. (32) Kyte, J.; Doolittle, R. F. J. Mol. Biol. 1982, 157, 105-132.
6276 Analytical Chemistry, Vol. 79, No. 16, August 15, 2007
Missed cleavages were most commonly observed in TA10-11 and TA20-21. According to the same index, the tryptic cleavages sites of these peptides and TA 14-17 (all arginines) are located in the most polar part of the A-chain. This indicates that polar regions may be more resistant to solvent-assisted trypsin digestion than other regions of the protein, in accordance with findings by Russell et al.20 indicating that repeated lysine residues in protein chains are strongly resistant to methanol-assisted trypsin digestion. In ricin, there are no sequences of lysines. However, in TA10-11 and TA20-21, arginine is situated close to the polar amino acids asparagine, aspartate, and glutamine. These regions seem to demonstrate intermediate resistance, and a mixture of complete and partial cleavage was obtained (see Table 2). In solvent-assisted digestion, it seems that polar parts of the protein are the most critical, and it was observed during the method optimization that increasing the organic solvent content favored digestion of problematic sites. However, in the optimized procedure, missed cleavages were shown to be a minor problem. It was also noted that intact disulfide bridges did not cause any problems in this procedure and peptides containing disulfide bridges were detected at adequate intensities. Verification of Ricin-B-Chain Tryptic Peptides. In the B-chain, 16 out of 20 potential tryptic fragments were verified (Table 2). Similarly to chain A, there are several tryptic fragments with sequence differences to RCA in chain B, notably two disulfidelinked peptides, TB3-ss-TB5 and TB14-ss-TB16 and TB10, with an intact internal disulfide bond. It is interesting to note that the disulfide bonds do not interfere with digestion even though the tryptic site is close or adjacent to the cysteine. Fragments identical to RCA are TB2 and TB13. The recovered peptides correspond to 71% of the B-chain amino acid sequence. The short fragments TB4,
TB7, and TB9 each contain 1-4 amino acids and were not recovered. TB12 is a large peptide, 66 amino acids long (Mw 6934.3). In addition, it is glycosylated and has one internal cysteine disulfide bond. No response was detected from TB12, either following organic solvent-assisted trypsin digestion or following the reduction-alkylation strategy. Glycosylation in TB11 was confirmed by the presence of diagnostic carbohydrate fragment ions in the product ion spectrum of the triply charged molecular ion at m/z 997.8. This was consistent with the results following the reduction-alkylation strategy.17 Partially missed cleavages were noted in TB6-7 and TB1718, supporting the discussion above that those sequences with more polar amino acids are more resistant toward solvent assisted trypsin digestion. HPLC-ESI MS/MS Screening of Crude Toxin Extracts. Scope of the Method. This method was developed to meet the need to analyze samples such as powders contained in threatening letters for the presence of protein toxins, ricin in particular. Such samples may be of a limited size, placing high demands on the sensitivity of the instrumentation. In a sample consisting of unpurified, homogenized castor beans, the ricin content could be ∼1%. Starting with 10 mg of ground seeds, extraction with 100 µL of dilute acid would provide sufficient sample for ∼25 injections for LC-MS analysis, corresponding to ∼60 pmol of ricin/injection, and even though the most easily detected peptides will require only 1-5 pmol to produce an intense peak with the standard electrospray source used, such “sample overload” will assist the analysis of low-intensity peptides. Greater sensitivity could be obtained by using a nanospray-ESI source, but the standard source was considered adequate for this purpose. The components detected by LC-ESI MS analysis of a digested crude extract was dominated by doubly and triply charged peptides from ricin and from other acid-extractable proteins. Examples of such proteins are RCA and 2S albumin.17 Multicharged components of charge state 5 and higher, which could not be fragmented even at high collision energy, indicated the presence of proteins resistant to the presented digestion procedure. Since ricin-specific peptides may be minor components, data-dependent analysis directed by an include list of mass and charge state of these peptides is required in order to selectively extract their spectrum from the relatively complex mixture of other components. However, a live sample could contain a much higher concentration of ricin than a crude plant homogenate. In the analysis of crude extracts presented below, a sample size equivalent to ∼10 mg of seed homogenate was used, which corresponds to a “worst case” from the analytical perspective. Ricin. From a crude extract of the sample, ricin could be identified using product ion spectra of ricin-specific tryptic peptides. Most diagnostic peptides from the A-chain were detected using DDA (Figure 3A) and manually interpreted to confirm their identity. From the B-chain, DDA revealed the presence of all ricinspecific peptides except TB8, TB10, and TB12. Disulfide-linked fragments TB3-ss-TB5 and TB14-ss-TB16 were also identified. The presence of the forensically highly important interchain disulfidelinked peptide TA24-ss-TB1 was also verified (Figure 4). Abrin. The same analytical protocol was applied to homogenized seed material from A. precatorius in order to evaluate the method for another RIP-II protein with structure and function
Figure 3. Data-dependent analysis with automatic tandem analysis of potential toxin peptides. The inset chromatogram illustrates a survey scan. (A) DDA-MS/MS analysis of a crude plant extract containing ricin. (B) DDA-MS/MS analysis of a crude plant extract containing abrin.
similar to that of ricin.7,8 Four different varieties of abrin are found in Swissprot: abrin-a, -b, -c, and -d.23 (For consistency with the peptide notation used for ricin, subscripts A and B are used to designate the polypeptide chains A and B, respectively, and superscripts a, b, c, and d to designate the four isoforms of abrin, denoted abrin-a, etc., in Swissprot). Potential tryptic peptides of abrin-a, -b, -c, and -d were localized by comparing theoretical mass/charge values with data from the LC-MS peptide map. The identity of potential abrin peptides was verified by their product ion spectra (Supporting Information, Table 2). Of these peptides, three are found in other proteins related to Abrus seeds. TA15a is also found in A. precatorius agglutinin,33 and two sequences, TA2b,c,d and TB10a,b/TB9c,d, are found in pulchellin, a toxin from the related Abrus pulchellus.34 However, it is unlikely that these peptides would (33) Liu, C. L.; Tsai, C. C.; Lin, S. C.; Wang, L. I.; Hsu, C. I.; Hwang, M. J.; Lin, J. Y. J. Biol. Chem. 2000, 275, 1897-901. (34) Ramos, M. V.; Mota, D. M.; Teixeira, C. R.; Cavada, B. S.; Moreira, R. A. Toxicon 1998, 36, 477-484. (35) Halling, K. C.; Halling, A. C.; Murray, E. E.; Ladin, B. F.; Houston, L. L.; Weaver, R. F. Nucleic Acids Res. 1985, 13, 8019-8033. (36) Lamb, F. I.; Roberts, L. M.; Lord, J. M. Eur. J. Biochem. 1985, 148, 265270. (37) Roberts, L. M.; Lamb, F. I.; Pappin, D. J.; Lord, J. M. J. Biol. Chem. 1985, 260, 15682-15686. (38) Roepstorff, P.; Fohlman, J. Biomed. Mass Spectrom. 1984, 11, 601.
Analytical Chemistry, Vol. 79, No. 16, August 15, 2007
6277
Figure 4. Verification of the forensically highly important presence of the interchain disulfide-linked peptide TA24-ss-TB1, by the product ion spectrum from data-dependent analysis. Precursor ions were automatically selected from an include list of ricin- and abrin-specific tryptic peptides.
be found in a pulchellin tryptic digest, since the amino terminals are preceded by glutamate and glutamine, respectively, not lysine or arginine. Using DDA, most diagnostic peptides from the A-chain were verified and are shown in Figure 3B. In addition, DDA, and corresponding product ion spectra, identified two B-chain peptides, TB19a,b,d/TB18c, and TB27b/TB26c (where the backslash, /, indicates an identical amino acid sequence). The A-chain of abrin-b, -c, and -d isoforms are represented by only two peptides common to more than one of the isoforms. Programming for MS/MS switching between time windows for preselected marker peptides with known retention times often provides greater sensitivity than DDA. An additional LC-MS/MS analysis targeting specific tryptic peptides provided product ion spectra from [TB6b-ss-TB8b]+3, [TB10a,b/TB9c,d]+2, [TB26a/TB24d]+3, and [TB27b/TB26c]+2 and, thus, additional evidence regarding the B-chain that assisted in the identification of the abrin isoforms present. Identification of RIP II Toxins. In a previous paper, we suggested that an unambiguous identification of a protein toxin should include MS/MS data from at least two unique peptides from each polypeptide chain.17 In the case of RIP II toxins, this implies two peptides from each of the A- and B-chains. DDA analysis based on survey scans and automatic MS to MS/MS switching on candidate marker peptides specified in the include list provided sufficient evidence for the presence of ricin and abrin from comparable sample amounts. Information on abrin isoform distribution could be obtained from a second MS/MS analysis of the sample by preprogramming for isoform-specific peptides. However, unambiguous identification of all four isoforms in this sample was not possible. Intact disulfide bonds are often not observed since reduction and alkylation is normally performed to make protease-resistant proteins amenable to enzymatic digestion. The procedure also facilitates interpretation of the product ion spectra of the resulting peptide derivatives. In addition, when screening for targeted toxins, disulfide bonds could provide diagnostic information and indications of an intact active protein. In particular, the disulfide bond between ricin chains A and B is important for the activity of the toxin and is characterized, for ricin, by the tryptic fragment [TA24-ss-TB1]. This disulfide-linked peptide is also found in RCA, but its presence, together with ricin-specific peptides and other intact disulfide-linked peptides from the B-chain, is strongly
6278 Analytical Chemistry, Vol. 79, No. 16, August 15, 2007
indicative of an intact link between ricin A- and B-chains. Thus, this information will strengthen evidence of possible violations of the CWC.12 Taken together with ricin-specific information from peptides with intact disulfide bonds (TB10, [TB3-ss-TB5], [TB14ss-T B16]), we suggest that the LC-ESI-MS/MS analysis provides sufficient evidence of an active protein and does not need complementary MALDI-MS or in vitro toxicity analysis. Abrin has corresponding disulfide bridges, but only the B-chain disulfide bound peptide [TB6a-ss-TB8a] was identified in our analysis. The remaining disulfide bonds in abrin are found in peptides larger than 3200 Da that are probably discriminated against in the ESI analysis due to their size. This possibility will be investigated further in future work. CONCLUSIONS The method provides rapid, unambiguous identification of ricin. We believe that requirements for unambiguous identification of ricin should include MS/MS data on at least two peptides specific for each of the ricin chains in order to meet stringent requirements for forensic analysis and analysis made in connection with the Chemical Weapons Convention. In addition, the method leaves disulfide bonds intact and provides information about the linkage between the two chains. This is considered to be key information regarding ricin in an OPCW verification context. The same protocol can also be used for verification of abrin, another highly toxic RIP II protein. However, an intact disulfide bond between the A- and B-chains in abrin could not be demonstrated. The use of an organic solvent to assist trypsin digestion dramatically shortens the sample preparation time, from an overnight digestion in the standard procedure to a 1-h digestion. Thus, the response time for an unambiguous answer regarding the presence or absence of ricin in a sample is shortened from about 24 to 2 h. SUPPORTING INFORMATION AVAILABLE Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review January 29, 2007. Accepted May 30, 2007. AC0701740
