Detection and Quantification of Ricin in Beverages ... - ACS Publications

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Detection and Quantification of Ricin in Beverages Using Isotope Dilution Tandem Mass Spectrometry Sara C. McGrath,† David M. Schieltz,† Lisa G. McWilliams,‡ James L. Pirkle,† and John R. Barr*,† †

Division of Laboratory Sciences, National Center for Environmental Health, Centers for Disease Control and Prevention, 4770 Buford Highway, Atlanta, Georgia 30341, United States ‡ Battelle (on Contract with the Division of Laboratory Sciences), 4770 Buford Highway, Atlanta, Georgia 30341, United States ABSTRACT: The toxic plant protein ricin has gained notoriety due to wide availability and potential use as a bioterrorism agent, with particular concern for food supply contamination. We have developed a sensitive and selective mass spectrometry-based method to detect ricin in tap water, 2% milk, apple juice, and orange juice. Ricin added to beverage matrices was extracted using antibodybound magnetic beads and digested with trypsin. Absolute quantification was performed using isotope dilution mass spectrometry with a linear ion trap operating in product-ion-monitoring mode. The method allows for identification of ricin A chain and B chain and for distinction of ricin from ricin agglutinin within a single analytical run. Ricin-bound beads were also tested for deadenylase activity by incubation with a synthetic ssDNA oligomer. Depurination of the substrate by ricin was confirmed by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOFMS). This method was used successfully to extract ricin from each beverage matrix. The activity of recovered ricin was assessed, and quantification was achieved, with a limit of detection of 10 fmol/mL (0.64 ng/mL).

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icin is a naturally occurring toxin produced by the seeds of the castor bean plant. Castor beans are common, and ricin is relatively easy to prepare, making the toxin a likely bioterrorism agent.1,2 The Centers for Disease Control and Prevention has identified ricin as a Category B agent, and ricin remains the only protein listed as a Schedule 1 chemical by the Chemical Weapons Convention.3,4 There is currently no antidote for ricin poisoning.5 Ricin is a protein composed of two distinct subunits, A chain and B chain. The B chain lectin will bind galactose-containing receptors on eukaryotic cell surfaces, initiating endocytosis of ricin by the cell. The A chain provides the deadenylase activity that irreversibly depurinates 28S rRNA and terminates protein synthesis within the cell.6
9 Ricin is produced in the castor bean along with the homologous protein Ricinus communis Agglutinin 120 (RCA120).10 RCA120 is composed of two A chains and two B chains with sequence homology to ricin A and B chains greater than 85%, but RCA120 has only a fraction of ricin’s toxic potency.11 Because of the similarity of RCA120 to ricin, and the ease with which the RCA120 monomers disassociate in vitro, the two proteins are very difficult to separate. Many methods currently in use for ricin detection cannot distinguish the two proteins.12 Because of the wide availability and relative stability of ricin, concerns exist that the food supply could be deliberately contaminated.13 One high-priority category is beverages commonly consumed by children. While ingestion of ricin is not the most potent route of intoxication,5 the danger does exist and significant effort has been put toward development of highly sensitive methods for detection of ricin in foods.12,14
23 Most r 2011 American Chemical Society

methods currently used for ricin detection fall into three categories: (i) methods that detect the presence of ricin through immunogenic interactions (such as enzyme-linked immunosorbent assay (ELISA);24,25 (ii) methods that exploit the enzymatic activity of ricin;8,26,27 and (iii) methods that detect the presence of castor bean DNA in a sample with the assumption that ricin toxin would also be present (such as polymerase chain reaction (PCR16)). These methods are generally fast, have a low limit of detection (LOD), and do not require expensive instrumentation. A table provided by Lubelli et al. summarizes various techniques used for ricin detection with the corresponding LOD.28 However, none of the published methods represent a truly comprehensive assay. While PCR is sensitive and can be quantitative, it is ultimately an indirect test that does not measure the presence of the actual toxin. Measurements that rely on enzymatic activity can be confounded if other toxic proteins are present in the sample. ELISA could return false positive results if the antibodies used cross-react with similar proteins or bind nonspecifically. Some of the more sophisticated methods require more than 24 h to determine a result. Methods that use mass spectrometry for ricin analysis have been developed in recent years. Tryptic digestion and mass spectrometry methods have been used to identify, though not quantify, peptides unique to the ricin protein.29
32 Liquid chromatography
mass spectrometry (LC
MS) quantitation of adenine Received: September 29, 2010 Accepted: March 1, 2011 Published: March 23, 2011 2897

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Analytical Chemistry released by ricin is sensitive, but the LOD is limited by background contamination from naturally occurring adenine.33,34 Only one method published in the literature combines a measurement of ricin enzymatic activity with proteomic identification of the toxin, although quantitation of the amount of toxin present or correlation of the quantity of toxin with the enzymatic activity detected was not reported.21 Many methods use antibody capture as part of the analysis so that the method can be applied to complex matrices. Antibody purification followed by mass spectrometric quantification has been demonstrated in biomarker discovery and validation, and this strategy shows high specificity and good linear dynamic range.35
38 The method presented here uses mass spectrometry in a two-pronged approach for detection and quantification of ricin: LC/product ion monitoring (PIM) MS for absolute quantification of toxin, combined with an enzymatic assay and matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOFMS) detection to determine functional activity. Absolute quantification of ricin is achieved through use of isotope dilution mass spectrometry (IDMS) and a linear ion trap mass spectrometer. The term “absolute quantification” refers to measuring the quantity of a substance against a known standard using a calibration curve and is used here to differentiate from relative quantification. The term “relative quantification” is generally used when the quantity of one substance in a sample is compared to the quantity of the same substance in a second sample and does not require that the exact amount of the substance be known. The concept of IDMS, as applied to protein quantification, presumes the quantity of a peptide measured by the mass spectrometer is representative of the quantity of whole protein present in a sample.39 Basic method design involves tryptic digestion of the target protein and identification of reliable peptide digest fragments. A synthetic heavy isotope peptide internal standard, differing from the native target only by stable isotopes of carbon and/or nitrogen, will have chromatographic retention times and ionization efficiencies identical to the native. The two peptides can be captured within the same chromatographic time window, and the resulting mass spectrometric ion intensities can be directly compared. The ratio of the chromatographic peak area of the native peptide to the chromatographic peak area of the internal standard is used to generate a calibration curve and quantify the amount of protein in an unknown sample.35,37,40 The mass spectrometric measurement of ricin’s deadenylase activity used here was developed on the basis of assays described in the literature.21,27,41 Ricin is incubated with a synthetic DNA mimic of the toxin’s natural rRNA target, and the product of the reaction is detected by MALDI-TOFMS. The immunopurification step prior to MS analysis allows the method to be applied to complex matrices contaminated with ricin. This method was designed to provide the best quantitative measurement of ricin while retaining the top features of existing assays. Ricin can be quantitatively recovered from complex matrices with a limit of detection (LOD) as low as 5 fmol (320 pg) per 0.5 mL of sample. The specificity of the linear ion trap mass spectrometer is exploited so that both ricin A chain and B chain are positively identified in a single experiment. Ricin deadenylase activity is quickly and accurately determined by MALDI-TOF detection of the DNA oligomer reaction product, rather than the released adenine. Because the final quantitative measurement is made by the linear ion trap, the false positive rate of the analysis is not dependent on highly specific antibodies in

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the immunopurification step. PIM analysis on the linear ion trap excludes from the analysis any other proteins that may cross-react with the antibodies or are copurified with ricin. The LD50 for human ricin ingestion is estimated at 3 mg/kg or 105 mg (1.6 μmol) for a 35 kg child.5,42 In an 8 oz beverage, a deadly ricin concentration is approximately 6.5 nmol/mL. The method described here has been applied to beverages that have been spiked with ricin or castor beans, with ricin quantified 100 000fold lower than the estimated LD50.

’ MATERIALS AND METHODS Safety Considerations. Ricin is a potent toxin, and all experiments described here using ricin or castor bean extracts were performed in a level-2 biosafety cabinet with a HEPA filter. After tryptic digestion, samples containing ricin are no longer toxic and may be handled safely using standard laboratory practice. Preparation of Magnetic Beads. Biotinylated anti-ricinus communus agglutinin polyclonal antibodies were purchased from Vector Laboratories (Burlingame, CA) in 5 mg lyophilized aliquots and reconstituted to 1 mg/mL in doubly distilled water. These antibodies were raised against the ricin holotoxin and were expected to cross-react with RCA120. MyOne T1 streptavadincoated magnetic beads were purchased from Invitrogen (Carlsbad, CA). The magnetic beads are 1 μm in diameter and are sold as a slurry of approximately 10 mg (7
10  109 beads) per mL. Beads were prepared for binding to antibodies according to manufacturer’s instructions. Briefly, 75 μL of 1 mg/mL biotinylated polyclonal ricin antibodies were combined with 500 μL of magnetic beads and 4 mL of phosphate-buffered saline (PBS); the mixture was incubated for 1 h at room temperature with endover-end rotation. After 1 h, the supernatant was removed and the beads were washed twice with PBS. Beads were resuspended to 500 μL with PBS containing 0.05% tween20 (PBST 0.05%) and were ready to use immediately or could be stored for up to 4 weeks at 4 °C. The concentration of beads in the slurry does not change after conjugation of the beads to antibodies. The manufacturer estimates a maximum binding capacity of 20 μg of biotinylated antibodies per 1 mg of beads. Recovery of Ricin and RCA120 from Matrix. Purified ricin (Ricinus communis agglutinin II) and purified RCA120 agglutinin were purchased from Vector Laboratories as a 5 mg/mL solution. Toxin and agglutinin spiked into sample matrices were allowed to incubate for 30 min and then were recovered using prepared capture beads in an automated fashion using the Kingfisher96 magnetic particle processor (Thermo Scientific, Waltham, MA). Each experiment used 15 μL of ricin capture beads constantly agitated for 1 h in 0.5 mL of ricin-containing sample solution. At the end of 1 h, the capture beads were removed from the sample solution by the particle processor and washed four times: once in 1 mL of PBST 0.05%, once in 1 mL of PBST 0.01%, once in 0.5 mL of PBS, and once in 200 μL of distilled water. Capture beads were then eluted in 100 μL of distilled water. Ricin capture experiments were performed with ricin extracted from PBST 0.05% and four beverage matrices: tap water, 2% milk, apple juice, and orange juice. All beverage items were purchased from a local grocery store. Castor Bean Extraction. Castor beans (variety Gibsonii) purchased from Horizon Herbs (Williams, OR) had approximately the same weight (462 mg (1.5%). For extraction experiments, whole beans were ground roughly with a stone tissue grinder (FisherScientific, Pittsburgh, PA) and mixed with 50 mL of 2898

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Analytical Chemistry

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Table 1. Ricin Tryptic Peptides and Transitions Used for Quantitation peptidea, protein, chain

peptide sequenceb, c

peptide ion [m/z]2þ

product ion [m/z]

HEIPVLPNR

537.81

b3 [380.2]þ, y6 [695.4]þ, y7 [808.5]þ, y8 [937.5]þ

HEIPVL[P*]NR

541.06

b3 [380.2]þ, y6 [701.5]þ, y7 [814.7]þ, y8 [943.4]þ

VGLPINQR

448.77

y5 [314.2]2þ, y6 [370.8]2þ, y5 [627.3]þ, y7[797.5]þ

VGLPIN[Q*]R

452.41

y5 [317.8]2þ, y6 [374.8]2þ, y5 [634.4]þ, y7 [804.5]þ

VGLPISQR

435.26

y5 [300.7]2þ, y6 [357.4]2þ, y5 [600.3]þ, y7 [770.5]þ

VGLPIS[Q*]R

438.91

y5 [304.7]2þ, y6 [360.7]2þ, y5 [607.4]þ, y7 [777.4]þ

T10 Ricin, A

YTFAFGGNYDR

655.79

y6 [681.3]þ, y7 [828.4]þ, y8 [899.5]þ, y9 [1046.5]þ

T11 Ricin, A

YTFAFGGNYD[R*] LEQLAGNLR

660.79 507.29

y6 [691.3]þ, y7 [838.4]þ, y8 [909.4]þ, y9 [1056.5]þ y4 [459.3]þ, y5 [530.4]þ, y6 [643.4]þ, y7 [771.5]þ

LEQLAGNL[R*]

512.29

y4 [469.4]þ, y5 [540.4]þ, y6 [653.4]þ, y7 [781.5]þ

NDGTILNLYSGLVLDVR

931.50

y8 [858.6]þ, y9 [1021.6]þ, y11 [1248.8]þ, y12 [1361.7]þ

NDGTILNLYS[G*]LV[L*]DVR

936.60

y8 [868.6]þ, y9 [1031.6]þ, y11 [1258.8]þ, y12 [1371.7]þ

NDGTILNLYNGLVLDVR

945.01

y8 [885.6]þ, y9 [1048.5]þ, y11 [1275.7]þ, y12 [1388.8]þ

NDGTILNLYN[G*]LV[L*]DVR

950.19

y8 [895.6]þ, y9 [1058.5]þ, y11 [1285.7]þ, y12 [1398.8]þ

T6 Ricin, A and RCA120, A T7 Ricin, A T7 RCA120, A

T18 Ricin, B T18 RCA120, B a

Trypsin digest peptides are numbered from the protein N-terminus. b Underlined residues indicate sequence differences between ricin and RCA120. c Residues indicated with [*] have been fully labeled with 13C and 15N.

beverage matrix. The bean mash was allowed to incubate in the matrix for 18 h, then the solution was centrifuged at 3700 rpm for 5 min to settle large particles. After centrifugation, a 0.5 mL aliquot of supernatant was extracted with antibody-coated beads and analyzed according to the method described. Protein Digest. Rapigest SF surfactant has been shown to enhance the tryptic digestion of ricin.43,44 Rapigest SF (Waters Corporation, Bedford, MA) was purchased in 1 mg lyophilized aliquots and reconstituted to 0.1% in 100 mM ammonium bicarbonate. Promega Trypsin Gold (mass spectrometry grade, Fisher Scientific) purchased as 100 μg of lyophilized enzyme was reconstituted to 1 μg/μL in 50 mM acetic acid. Digest experiments were conducted by buffer-exchanging ricin-containing magnetic beads into 15 μL of 0.1% Rapigest SF solution. Samples were heated to 100 °C in a dry heat block for 10 min and then cooled to room temperature before adding calcium chloride (final concentration 1 mM) and 1 μL of trypsin solution. A high molar ratio of trypsin-to-ricin (about 8:1) and incubation for 1 h at 37 °C were empirically determined to provide the best digestion of the toxin.40 The tryptic digest reaction was quenched and Rapigest SF was degraded by the addition of 2 μL of 1 M HCl to the samples and incubating them in a 37 °C water bath for 1 h. Digested ricin and RCA120 samples were prepared for LC
MS analysis by centrifuging for 1 min at 14 500 rpm and removing all liquid supernatant to an autosampler vial. Preparation of Peptide Standards. Ricin quantification peptides were chosen to represent both the A and B chains of ricin and also to distinguish ricin from RCA120 where possible (Table 1). Peptides that are glycosylated as well as those containing cysteine, methionine, or tryptophan were avoided. In total, 7 peptides were selected for synthesis by Midwest Biotech, Inc., (Fishers, IN). Peptides were aliquotted in-house using a Beckman Coulter Biomek NXP Laboratory Automation Workstation (Fullerton, CA), and amino acid analysis was performed by Midwest Biotech, Inc. on 5 randomly selected aliquots from each peptide. The amino acid analysis results for each peptide agreed with a coefficient of variance (CV) of less than 4%. Working stock solutions were generated by reconstituting individual peptide aliquots in 0.1% formic acid (v/v) to 5 pmol/μL using the method described previously.44 Heavy isotope internal standard peptides were

combined in equal molar amounts and diluted to 100 fmol/μL. For quantification experiments, 100 fmol of internal standard solution was added to each sample before analysis. Liquid Chromatography. Chromatographic separation was performed using an Agilent 1200 capillary pump (Agilent Technologies, Santa Clara, CA) equipped with a Magic C18 AQ reversedphase analytical column (150 mm  1.0 mm i.d., 5 μm particle size, 200 Å pore size, Michrom Bioresources, Inc., Auburn, CA) operating at 80 μL/min. Mobile phases were A = water with 0.2% formic acid and 0.005% trifluoroacetic acid and B = acetonitrile with 0.2% formic acid and 0.005% triflouroacetic acid. All solvents were HPLC grade Honeywell Burdick and Jackson and purchased from VWR (West Chester, PA). Samples were introduced at 5% B, and elution was achieved using a sawtooth gradient.45 In phase 1, the gradient was increased from 5% B to 15% B over 5 min. The gradient was decreased to 11% B over 1 min and held constant at 11% B for 1 min. In phase 2, the gradient was increased from 11% B to 17% B over 5 min. The gradient was decreased to 15% B over 1 min and held constant at 15% B for 1 min. In phase 3, the gradient was increased from 15% B to 90% B over 4 min. The gradient was held constant at 90% B for 3 min and then decreased to 5% B over 2 min. A time of 15 min was required for column re-equilibration. The LC system was equipped with a 40 μL sample loop to allow the entire sample aliquot to be injected at once. Mass Spectrometric Quantification. Mass spectrometric quantification was performed using the LTQ module of an LTQ-Orbitrap XL (Thermo Scientific, San Jose, CA). Samples were introduced to the LTQ by electrospray ionization (ESI) operating in positive ion mode. Instrument parameters were as follows: sheath gas flow rate 20, spray voltage 4 kV, capillary temperature 275 °C. The LTQ was operated in product ion monitoring or PIM mode,46 where a list of precursor ions entered into the method directed the LTQ acquisition. Precursor ions selected correspond to doubly charged, monoisotopic peptide ions for both the native and the internal standard peptides. Collision-induced dissociation (CID) was triggered when one of the listed precursor ions was detected by the LTQ. The mass selection window was set to (0.75 Da with a qz = 0.25 and a normalized 2899

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Analytical Chemistry collision energy of 35 for each peptide. All product ions within a mass range from the [M þ 2H]2þ of the parent ion to the low-mass cutoff (determined by the LTQ for a qz of 0.25) were acquired during CID, so that full tandem mass spectra from each parent ion were recorded. Extracted ion chromatograms (XIC) indicating the temporal location of specific parent ion-to-product ion transitions were created using filters applied in the processing mode after acquisition. The filter allows only parent ion-toproduct ion transitions specified by the operator to be shown in the XIC. Four transitions were monitored for each peptide, and the absolute intensities of the product ion signals were summed to generate an XIC for each peptide. Because of the specificity of PIM detection, only one peak appeared in the XIC after the data had been filtered. Parent and product ion m/z values used in creating the acquisition methods and filters are listed in Table 1. Peak areas calculated from the XIC for native and internal standard calibration peptides were used to construct a calibration curve for standards and to determine the amount of ricin in an unknown sample. Activity Assay. Ricin was tested for depurination activity using a short DNA substrate designed to mimic ricin’s natural rRNA target.47 The DNA substrate GCGCGAGAGCGC synthesized by Integrated DNA Technologies (Coralville, IA) was received as lyophilized material and was reconstituted to 1 nmol/μL in doubly distilled water. For the activity assay, ricin or ricin-bound magnetic beads were buffer-exchanged into 20 μL of reaction buffer (10 mM ammonium citrate, 1 mM ethylenediaminetetraacetic acid, pH 4) and 5 μL of 500 pmol/μL DNA substrate was added. Reactions were allowed to proceed at 37 °C for 4
24 h. MALDI-TOF Analysis. MALDI matrix was a saturated solution of 3-hydroxypicolinic acid (>99.0% puriss, SigmaAldrich, St. Louis, MO) in 50:50 deionized water
acetonitrile with ammonium tartrate and ammonium citrate added to a final concentration of 10 mM. At the end of the activity assay reaction time, 2 μL of reaction buffer was removed and added to 18 μL of MALDI matrix solution. Samples were spotted in triplicate (0.5 μL per spot) on a steel MALDI plate and analyzed with a 4800 Plus Proteomics Analyzer MALDI-TOF
TOF (Applied Biosystems, Foster City, CA). Data were acquired in linear positive ion mode using a nitrogen laser at 337 nm. Each spectrum represents a total of 3600 laser shots. Ricin in this analysis has not been deactivated and remains extremely toxic, and the elevated safety considerations previously mentioned should be observed.

’ RESULTS AND DISCUSSION IDMS Quantification of Ricin. Many peptide and protein quantification methods use triple quadruple mass spectrometers (QqQ) operating in multiple-reaction-monitoring (MRM) mode, but the LTQ mass spectrometer provides similar information and offers some advantages over the QqQ. In particular, the LTQ records the complete product ion spectrum from CID, whereas the QqQ acknowledges only specific parent-to-product ion transitions. Having a complete product ion spectrum for each parent ion can be useful when isobaric interferences are present or when the analyte quantity is low. An additional advantage is that LTQOrbitrap instruments are commonly used in laboratories for proteomic discovery work, and the ability to develop quantification methods on the same instrument is convenient and costeffective.36,38 Ricin toxin polyclonal antibodies are available commercially and have sufficient specificity for this method to be

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effective, because ultimately the LTQ is used for distinguishing peptides in the mixture. Use of an antibody purification step prior to digestion and LC
MS allows the method to be applied to complex matrices without compromising detection limits, while simultaneously concentrating the amount of toxin in a large sample and improving sensitivity. Choice of peptides for quantification is a crucial step in IDMS method development.36
38 A total of five peptides were chosen for quantification empirically from tryptic digests of purified ricin analyzed in data-dependent mode by the LTQ-Orbitrap. The candidate peptide sequences were subjected to alignment using the BLAST algorithm (http://blast.ncbi.nlm.nih.gov/Blast.cgi) to ensure that the peptides would unambiguously identify their respective proteins (protein database, SwissProt; algorithm, Blastp; maximum target sequences, 100; algorithm was set to automatically adjust for short input sequences; expect, 10; word size, 3; matrix, PAM30; gap costs, Existance 9, Extenstion 1). Good candidate peptides were detected with high sensitivity as an [M þ 2H]2þ ion, dissociated easily via CID, and could be separated by reversed-phase liquid chromatography. Tryptic peptides containing residues likely to undergo chemical modification during sample handling (i.e., Cys, Trp, Met) were avoided.35,37,48 Although detection of one unique peptide is theoretically enough to identify ricin in a mixture, including four additional peptides in the analysis as confirmation ions makes the identification unambiguous.48 These peptides also allow ricin to be distinguished from RCA120, as well as confirm the presence of both A and B chains. The native tryptic peptides and corresponding internal standard peptides chosen for quantification are listed in Table 1, with residues containing 13C and 15N indicated by [*]. The method performance was evaluated by assessing quantification of ricin from a tryptic digest. Key figures of merit were linearity, dynamic range, reproducible area ratio of calibration standards, and the lowest peptide standard that could be accurately quantified. For this assessment, a protein calibration curve was generated using purified ricin toxin serially diluted from 5000 to 0.5 pmol/mL in PBST 0.05% (11 standards). This concentration of toxin kept the total digest volume low while maintaining a range of 10 000 to 1 fmol total protein injected onto the column. For each calibration standard, 2 μL of protein solution was added to the Rapigest solution and digested with trypsin. All samples were combined with 100 fmol of heavy-isotope peptide internal standard solution and analyzed by LC
IDMS in PIM mode. Of the five tryptic peptides considered for quantification, the T7 peptide from ricin was empirically found to yield an intense doubly charged ion and provide the best sensitivity in the analysis. The other three peptides unique to ricin (T10, T11, and T18) yield similar quantitative results to T7 when intact ricin is used as a calibration standard. With the use of T7 as a quantification peptide, the LOD for a digest of ricin toxin was 1 fmol protein injected on column. The calibration curve for this direct ricin digest dilution series had a linear response by LC
IDMS from 10 000 to 1 fmol protein injected on column, with an R2 value of 0.999. A second assessment of the analytical performance considered the effects of the immunopurification steps on the overall LOD. Because 0.5 mL of sample is used in immunopurification, the calibration curve samples were adjusted to maintain a range of 10 000 to 1 fmol protein injected on column. The protein calibration curve was generated using purified ricin toxin serially diluted from 20 000 to 2 fmol/mL (11 standards) in PBST 0.05%. The 2900

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Analytical Chemistry calibration curve samples were incubated with antibody-coated beads, and then the beads were washed using the Kingfigher96 to remove nonspecific binding and digested with trypsin. All samples were combined with 100 fmol of heavy-isotope peptide internal standard solution and analyzed by LC
IDMS in PIM mode. With the use of T7 as the quantification peptide, the LOD for ricin captured from PBST is 10 fmol/mL or 5 fmol of captured protein. The calibration curve for antibody-captured ricin had a linear response by LC
IDMS from 10 000 to 10 fmol/mL, with R2 value of 0.997. The increase in LOD from 1 fmol of protein to 5 fmol of protein was expected because the additional sample handling steps cause some loss of sample before analysis by the mass spectrometer. The decrease in dynamic range from 10 000 fmol protein to 5 000 fmol protein likely represents a binding capacity or binding equilibrium issue with the antibody-bound beads used in the immunopurification. Differentiation of Ricin and RCA120. As mentioned previously, ricin shares greater than 85% sequence homology with the less toxic agglutinin protein RCA120, and there are no known antibodies that can preferentially bind one protein over the other. While mass spectrometry proteomic analysis can easily distinguish the two on the basis of protein sequence, there are few tryptic peptides that are appropriate for quantitative measurement using LC
IDMS. The T7 peptide from ricin is one that can be used to distinguish ricin from RCA120 on the basis of amino acid sequence. To show that LC
IDMS can accurately quantify ricin even in the presence of RCA120, ricin and RCA120 were combined in PBST 0.05%, immunopurified, and quantified from the resulting tryptic digest using the ricin T7 peptide. The quantity of ricin spiked into each sample was held constant at 2 pmol protein, and the quantity of RCA120 was 2, 1, or 0.5 pmol protein. All samples were combined with 100 fmol of heavy-isotope peptide internal standard solution and analyzed by LC
IDMS in PIM mode. Results are shown in the first row of Figure 1. For each sample, the quantity of ricin recovered remained the same (within 7%), regardless of the amount of RCA120 present in the sample. This consistency indicates that only ricin is being quantified and that RCA120 is not contributing to the perceived amount of protein detected. However, RCA120 can be quantified when its corresponding T7 peptide is included in the LC
PIM analysis. Figure 1, middle row, shows the results when the above data are reprocessed using the T7 peptide from RCA120 for quantification. The amount of peptide detected decreases as the quantity of RCA120 in the samples decreases (2 to 0.5 pmol) and recoveries match the expected values within 6% for the three levels of RCA120. Use of the RCA120 T7 peptide in the LC
IDMS method allows accurate quantification of RCA120, and the presence of ricin in the mixture does not affect the quantity of RCA120 recovered. The ability to use peptides corresponding to ricin, RCA120, or both in a single analysis represents a significant advantage over other ricin detection methods. Ricin can be quantified independently from, or in combination with, RCA120 by choosing the quantification peptide specific for an application. To show that the Vector polyclonal antibody has equal affinity for ricin and RCA120, the data from the above experiment were reprocessed and quantified using the T6 tryptic peptide. The T6 peptide is conserved between ricin and RCA120, and quantification using T6 is independent of the identity of the molecule. The expected protein quantity was calculated to be the sum of the quantity of ricin and the quantity of RCA120 in each sample (4, 3, and 2.5 pmol, respectively.)

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Figure 1. Quantification of ricin and RCA120. Each sample contained 2 pmol of ricin, and the amount of RCA120 was 2, 1, or 0.5 pmol. The ratio of RCA120
ricin is denoted on the y-axis. Front row, quantification using ricin T7 peptide; middle row, quantification using RCA120 T7 peptide; back row, quantification using conserved T6 peptide.

When the T6 peptide is used for quantification, the amount of protein recovered for all three samples meets the expected ratio for a combination of the two proteins (Figure 1, back row). Because both ricin and RCA120 are recovered with similar efficiency when the proteins are analyzed either separately or together, we conclude that the polyclonal antibodies used for immunopurification have equal affinity for the two proteins. The LC
IDMS method can also be applied to determine the amount of RCA120 present as a contaminant in a sample of purified ricin. Ricin and RCA120 are very hard to physically separate from each other in a castor bean extract, and a small amount of RCA120 can be expected in a purified ricin sample. The quantity of RCA120 in a sample of purified ricin was determined using 2 pmol of Vector ricin digested with trypsin, combined with heavy-isotope peptide internal standard solution, and analyzed by LC
IDMS against a synthetic peptide standard curve. By comparing the results from ricin T7 and RCA120 T7, we calculated that the standard ricin material we obtained from Vector (lot U0718) contains 1.1 ( 0.2% of RCA120. Detection of Ricin in Beverages. Contamination of food with ricin or castor beans has been identified as a potential public health concern. Therefore, a comprehensive ricin detection method must be able to accurately and quantitatively detect ricin in food matrices. To test the robustness of the method, a panel of four beverages were spiked with purified ricin and analyzed by LC
IDMS. After incubation of ricin in each beverage for 30 min, a 0.5 mL aliquot of beverage was immunopurified, digested with trypsin, combined with heavy-isotope internal standard solution, and quantified by LC
IDMS using the ricin T7 tryptic peptide. For quantitation of unknown samples, a protein standard curve was generated from each beverage. Purified ricin was serially diluted from 20 000 to 2 fmol/mL (11 standards total) in the matrix of interest. Because 0.5 mL of sample was used in each analysis, this concentration maintains the desired calibration range of 10 000 to 1 fmol protein injected on column. Each set of protein calibration samples was immunopurified, digested with trypsin, and analyzed via LC
IDMS using the ricin T7 peptide. The resulting calibration curves were linear from 10 000 to 10 fmol/mL, with R2 values greater than 0.998. The LOD for each matrix is established at 10 fmol/mL (5 fmol protein on column). Matrix samples containing 4000 or 200 fmol/mL of ricin were assayed in triplicate alongside the standards and analyzed against the protein calibration curves (Table 2). The accuracy of 2901

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Table 2. Accuracy of Ricin from Different Matrices Quantified Using Ricin T7 Peptidea 200 fmol/mL

4000 fmol/mL

% accuracy

% cv

% accuracy

% cv

PBST

94.8

tap water

98.4

11.3

95.7

11.3

12.5

102.9

2% milk apple juice

13.2

113.3 95.0

7.1 6.0

104.8 97.6

8.3 2.6

orange juice

109.8

4.8

99.4

14.7

a

Protein standard curve extracted alongside unknowns. Experiments performed in triplicate.

ricin at the 4000 fmol/mL level was between 95 and 105%, and at the 200 fmol/mL level it was between 94 and 113%. The CV values were less than 15% overall, with several matrices less than 10% CV. Use of a protein calibration curve with the heavyisotope internal standard solution normalizes the test samples with the calibration samples, so that losses due to sample handling steps are experienced by all the samples. The in-matrix protein calibration curve compensates for variations in antibody capture of ricin and matrix effect interactions with the ricin protein. Quantitative values are therefore more representative of the true value of the samples. Incorporation of a protein standard curve with test samples results in a higher degree of accuracy and precision for quantification than is obtained by the use of the standard synthetic peptide curve. With the use of a peptide curve (purified calibration peptides diluted in PBST), recovery of ricin from beverages was as low as 29%, even from relatively simple matrices such as tap water. Performing an in-matrix protein calibration curve corrects for low recovery and allows for high accuracy in quantitating the amount of ricin present in a sample. Castor Bean Contamination of Beverages. The panel of four beverages was evaluated for ricin content after incubation with crushed whole castor using the LC
IDMS method. On the basis of the weight of beans used (∼450 mg) and literature reports of 1
5% toxin by weight,5 the maximum amount of ricin in an individual bean was estimated at 10 mg (100 nmol), well above the dynamic range of the LC
IDMS method. The LC
IDMS method has an upper limit of recovery of approximately 13 pmol/ mL based on the estimated amount of antibody bound to magnetic beads. To ensure that ricin recovered from matrices would fall within the established calibration range, tryptic digest samples were split into multiple aliquots before LC
IDMS analysis. For each beverage, a protein calibration curve was generated with purified ricin serially diluted from 20 000 to 2 fmol/mL (11 standards total) in the matrix. After serial dilution, the calibration standards were held at room temperature for 30 min before immunopurification, digestion, and analysis using LC
IDMS.

The amount of ricin recovered from each beverage was evaluated using both the common T6 peptide and the ricin T7 peptide. The quantity of ricin determined for the individual aliquots was summed to derive the final quantity of ricin listed in Table 3. Ricin was recovered from all of the beverages, although the amount of ricin recovered from each matrix varied. The most ricin was detected in the PBST sample, and the least amount in the orange juice sample (10 less than PBST), regardless of the quantification peptide used. Because the calibration curve samples were extracted from the same matrix as the unknown castor extract samples, factors such as low pH or matrix complexity should have had little effect on ricin recovery. However, it is possible that the 18 h incubation of castor bean mash with the beverage matrix could cause ricin to denature or become bound to other components in the beverage (such as lactose), thereby reducing the amount of ricin available to bind the magnet-bound antibodies. Because the samples contained crude castor extracts, a significant amount of RCA120 was expected to be present. The presence of RCA120 was indicated by the difference in quantitation values when the data were processed using ricin T7 peptide versus common T6 peptide. The amount of ricin as quantified by T7 was significantly less than that using T6, although the ratio of T7/T6 for each sample remained approximately the same (0.41 ( 4%). This may indicate that the amount of ricin in each castor bean was approximately the same. The exception to this pattern was the 2% milk sample, where only 27% of the T6 protein signal could be attributed to ricin. A possible explanation may be the high level of lactose in milk, which may preferentially bind ricin rather than RCA120 in solution. Test for Enzymatic Activity of Ricin. For analysis of ricin in food, it is important to know if the ricin is still enzymatically active. The assay described here determines the deadenylase activity of ricin with a DNA substrate that contains a GAGA tetraloop motif.47,49
53 The toxin removes the first adenine in the DNA tetraloop, resulting in a net loss of 118 Da from the mass of the substrate. The substrate and depurinated product can be detected by MALDI-TOFMS. While immunopurification is not necessary prior to the ricin activity test, it ensures that other enzymes or toxins that may be present in the sample do not contribute to the reaction product detected. Because our assay detects the depurinated DNA oligomer, not released adenine, there is less background contamination and positive results can be identified with more confidence. While this portion of the assay is not quantitative, the intensity of the depurinated DNA oligomer detected will increase with increasing amounts of ricin present. A MALDI-TOF spectrum of 500 pmol of DNA substrate is shown in Figure 2a, with the substrate [M þ H]þ observed at 3697 Da. Smaller peaks forming a cluster near [M þ H]þ correspond to [M þ Na]þ and [M þ K]þ ions. After incubation of 5 pmol of purified ricin with 500 pmol of DNA substrate for

Table 3. Quantity of Ricin Extracted from Different Matrices Incubated with Crushed Castor Beans castor bean mass (mg)

Common-T6 (pmol)a

PBST

466.2

110.2

41.0

0.37

tap water

455.7

50.7

23.7

0.47

matrix

a

Ricin-T7 (pmol)a

Ratio T7:T6

apple juice

454.9

38.8

15.4

0.40

2% milk

463.2

15.1

4.1

0.27

orange juice

469.3

10.2

4.7

0.42

Experiments performed in triplicate. 2902

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Figure 2. MALDI-TOF spectra of 500 pmol of DNA substrate reacted with ricin using the activity assay. DNA substrate ion is indicated with [M þ H]þ. DNA product of enzymatic reaction is indicated with a f. (a) DNA oligomer only, (b) activity of 5 pmol of neat ricin, (c) 5 pmol of ricin immunopurified from PBST, (d) 5 pmol of ricin immunopurified from tap water, (e) 5 pmol of ricin immunopurified from 2% milk, (f) 5 pmol of ricin immunopurified from apple juice, (g) crude ricin immunopurified from tap water, (h) crude ricin immunopurified from 2% milk, and (i) crude ricin immunopurified from apple juice.

4 h, the
118 Da reaction product can be observed at 3579 Da (Figure 2b). The signal for [M þ H
118]þ is low, and it is partially obscured by background peaks in the 3550
3620 Da range. The MALDI-TOF spectrum in Figure 2c shows the product formed from a 4 h reaction of 500 pmol of DNA substrate with 5 pmol of ricin that has been immunopurified from 0.5 mL of PBST 0.05% prior to the activity test. The signalto-noise has been increased 5 over that of the unbound ricin, and [M þ H
118]þ can now be clearly observed as a positive result. Ricin will continue to be enzymatically active so long as substrate exists in solution, so that longer reaction times will result in the formation of more depurinated product. However, the depurinated DNA product is somewhat unstable in the low pH reaction buffer and can degrade if the reaction proceeds for an extended length of time. The optimum reaction time was empirically determined to be between 4 and 6 h, and that time resulted in a detection limit of 1 pmol/mL immunopurified ricin when 500 pmol of DNA substrate is used. It should be noted that the detection limit for this MALDI-TOFMS assay is relatively high because the reaction solution must be diluted with MALDI matrix before analysis. After the initial 1:9 dilution, only 0.5 μL of the reaction/matrix solution is spotted on the MALDI plate and analyzed.

Enzymatic Activity of Ricin Recovered from Matrices. Ricin recovered from beverages, both purified toxin and extracts from castor beans, was tested to see if the ricin retained its deadenylase activity. Antibody-bound magnetic beads were used to extract ricin from beverages, then added to the reaction buffer with 500 pmol of DNA substrate and allowed to incubate for 4 h. MALDITOF spectra from activity reactions of 5 pmol of purified ricin recovered from 0.5 mL of tap water, 2% milk, and apple juice are shown in Figure 2d
f. The intensity of [M þ H
118]þ varies slightly among the three spectra, with the tap water sample showing the greatest [M þ H
118]þ intensity and the apple juice sample showing the least amount of [M þ H
118]þ intensity. While the MALDI-TOF data are not quantitative and the differences in intensity of [M þ H
118]þ are modest, it is likely that the [M þ H
118]þ intensity reflects the quantity of ricin extracted from each of the matrices by the antibody-bound beads. The beverages that were incubated with castor beans were also tested to determine the level of ricin activity retained. These matrices were incubated with crushed castor beans for 18 h before 0.5 mL aliquots were immunopurified, combined with 500 pmol of DNA substrate in activity buffer, and analyzed by MALDI-TOFMS. MALDI-TOF spectra from activity reactions of tap water, 2% milk, and apple juice incubated with crushed castor beans are shown in 2903

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Analytical Chemistry Figure 2g
i. Again, the intensity of [M þ H
118]þ varied according to the beverage, with the tap water showing the greatest [M þ H
118]þ intensity and the juices showing the least amount of [M þ H
118]þ intensity. After the activity test, the beads were digested with trypsin and analyzed using the LTQ to determine the amount of ricin present. The slight differences in enzymatic activity are reflected in the differences in the quantity of ricin determined by LC
IDMS (see Table 2).

’ CONCLUSIONS We present a quantitative analysis of ricin toxin using immuno-magnetic purification and isotope dilution mass spectrometry. With the use of this method, ricin can be quantified down to 10 fmol/mL in tap water, 2% milk, apple juice, and orange juice. Quantification is performed using heavy-isotope tryptic peptides and an LTQ-Orbitrap mass spectrometer operating in product ion monitoring mode. The method can be applied either to foods spiked with purified toxin or to those contaminated with crushed castor beans. The deadenylase activity of ricin can be assessed by MALDI-TOF mass spectrometric detection of a depurinated synthetic DNA substrate. The quantification assay and enzymatic activity assay can be run either in parallel (to return results faster when sample is plentiful) or the quantification can be performed on ricin-bound beads after the enzymatic activity has been assessed (when sample is limited). Analysis by mass spectrometry has many advantages over traditional molecular biology methods. Mass spectrometry provides a direct measure of the toxin itself, is not affected by interfering proteins, and has high sensitivity and selectivity. In addition, quantification can be achieved with high precision, even in the presence of complex matrices. While the LTQ itself is capable of distinguishing ricin from other proteins in a mixture, the immunopurification steps applied in the method described here help maintain a low detection limit and provide a second layer of specificity because antibodies selectively enrich ricin from a mixture. Prudent choice of tryptic peptides for IDMS detection allows quantification of ricin independently from RCA120. If peptides unique to RCA120 are used in the quantification method, that protein can also be quantified. Quantification of ricin is more accurate when the calibration curve samples are generated using purified toxin serially diluted in the same matrix as test samples. In this way, the calibration curve samples are subjected to the same sample handling procedures as the test sample and suffer the same matrix-dependent losses. When a protein calibration curve is used, high accuracy and precision were achieved for ricin quantification regardless of the beverage being tested. ’ AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected]. Phone: 770-488-7848. Fax: 770-4880509.

’ ACKNOWLEDGMENT Reference in this article to any specific commercial products, process service, manufacturer, or company does not constitute an endorsement or a recommendation by the U.S. government or the Centers for Disease Control and Prevention. The findings

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and conclusions reported in this article are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention.

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