Anal. Chem. 2006, 78, 6640-6644
Verification of Exposure to Organophosphates: Generic Mass Spectrometric Method for Detection of Human Butyrylcholinesterase Adducts D. Noort,* A. Fidder, M. J. van der Schans, and A. G. Hulst
Business Unit Biological and Chemical Protection, TNO Defense, Security and Safety, P.O. Box 45, 2280 AA Rijswijk, The Netherlands
We present a generic mass spectrometric method to verify exposure to organophosphates, based on the chemical conversion of the phosphylated peptides obtained after pepsin digestion of human butyrylcholinesterase (HuBuChE) to a common precursor peptide. After exposure of plasma to various organophosphates (nerve agents, pesticides), HuBuChE was isolated from plasma by procainamide affinity-based solid-phase extraction. Upon subsequent pepsin digestion, the respective phosphylated nonapeptides could be identified in the digests. After treatment of the pepsin digests with Ba(OH)2 in the presence of a nucleophilic tag (a thiol or amine), the phosphylated nonapeptides were transformed into a common tagged nonapeptide that could be analyzed sensitively by means of LC tandem MS. So far, best results were obtained with 2-(3-aminopropylamino)ethanol as nucleophilic tag. By applying the presented method, HuBuChE inhibition can now be monitored accurately by mass spectrometry, without advance knowledge of the structure of the inhibitor. Methods to verify exposure to organophosphate (OP) anticholinesterases, e.g., nerve agents, can play a pivotal role in case of a terrorist attack with nerve agents. In the same context, confirmation of nonexposure of worried citizens is of utmost importance. In recent years, we have developed basically two methods in order to detect exposure to nerve agents (for reviews, see refs 1-3). The fluoride reactivation method is based on the principle that upon incubation of phosphylated binding sites (e.g., inhibited human butyrylcholinesterase, HuBuChE, in plasma) with a large excess of fluoride ions, the phosphyl moiety is quantitatively converted into the corresponding phosphono- or phosphoro fluoridate.4 The latter can be isolated by solid-phase extraction and quantitated by GC-NPD or GC/MS. The other method is * To whom correspondence should be addressed. E-mail:
[email protected]. (1) Noort, D.; Benschop, H. P.; Black, R. M. Toxicol. Appl. Pharmacol. 2002, 184, 116-126. (2) Black, R. M.; Noort, D. In Chemical Weapons Convention Chemicals Analysis: Sample Collection, Preparation and Analytical Methods; Mesilaakso, M., Ed.; John Wiley & Sons: Chichester, UK, 2005; pp 403-431. (3) Noort, D.; Black, R. M. In Chemical Weapons Convention Chemicals Analysis: Sample Collection, Preparation and Analytical Methods; Mesilaakso, M., Ed.; John Wiley & Sons: Chichester, UK, 2005; pp 431-451. (4) Degenhardt, C. E. A. M.; Pleijsier, K.; van der Schans, M. J.; Langenberg, J. P.; Preston, K. E.; Solano, M. I.; Maggio, V. L.; Barr, J. R. J. Anal. Toxicol. 2004, 28, 364-371.
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based on mass spectrometric determination of specific nonapeptide adducts that result after pepsin digestion of modified HuBuChE.5 This method surpasses the limitations of the fluoride reactivation method since it can deal with HuBuChE inhibited with OPs that cannot be reactivated or that rapidly age (e.g., soman). One of the problems with both assays for assessment of OP exposure is that one has to know in advance which type of OP to screen for during mass spectrometric analysis. Therefore, a more generic mass spectrometry-based method for detection of phosphylated HuBuChE is highly desirable. In this respect, our attention was raised by a method that is used for the detection of phosphorylation sites in the field of proteomics.6-8 According to this method, proteins are treated with mild base in order to eliminate the phosphate function, resulting in the formation of a dehydroalanine residue in the protein. Subsequently, the formed dehydroalanine residue is scavenged with a simple or functionalized thiol or amine according to a Michael addition-type mechanism. We here report that this method, albeit in a slightly modified form, can be applied to OP-inhibited HuBuChE and that the result is irrespective of the agent used. EXPERIMENTAL SECTION Materials. Purified HuBuChE (EC 3.1.1.8) was obtained from Dr. B. P. Doctor of the Walter Reed Hospital (Washington, DC). Centrifugal ultrafilters (Centricon YM-3, 3 kDa or Amicon Ultra15, 100 kDa), were purchased from Millipore (Bedford, MA). Pepsin (EC 3.4.23.1) was purchased from Roche Diagnostics (Almere, The Netherlands). Sarin, soman, and VX were used from stocks within our laboratory. Dichlorvos was purchased from Chemservice (West Chester, PA). Human plasma was purchased from a blood bank (Sanquin, Leiden, The Netherlands). Ba(OH)2 was obtained from Riedel-de Haen. 1H,1H,2H,2H-Perfluorodecane1-thiol was purchased from Fluorous Technologies Inc. (Pittsburgh, PA). Ethanethiol, N-phenylethylenediamine, 2-phenylethanethiol, 3-(2-aminoethylamino)propylamine, 1,4-bis(3-aminopropoxy)butane, 2-(3-aminopropylamino)ethanol, benzylamine, (5) Fidder, A.; Hulst, A. G.; Noort, D.; de Ruiter, R.; Van der Schans, M. J.; Benschop, H. P.; Langenberg, J. P. Chem. Res. Toxicol. 2002, 15, 582590. (6) Oda, Y.; Nagasu, T.; Chait, B. T. Nat. Biotechnol. 2001, 19, 379-382. (7) Hathaway, G. M.; Zhou, J.; Rusnak, F. J. Biomol. Tech. 2002, 13, 228-237. (8) Knight, Z. A.; Schilling, B.; Row: R. H.; Kenski, D. M.; Gibson, B. W.; Shokat, K. M. Nat. Biotechnol. 2003, 21, 1047-1054. 10.1021/ac060954t CCC: $33.50
© 2006 American Chemical Society Published on Web 08/18/2006
Figure 1. Product ion mass spectrum of protonated molecular ion [M + H]+, 840.3 Da of FGE(S-ethylcysteine)AGAAS obtained after pepsin digestion of processed (Ba(OH)2/EtSH), purified HuBuChE.
Figure 2. Selection of nucleophiles used for modification of FGE(phosphoserine)AGAAS residues after base-catalyzed β-elimination of the phosphyl group.
1-(2-aminoethyl)piperidine, 2-mercaptoethylamine, and 1,3 bis(3aminopropyl)-1,1,3,3-tetramethyldisiloxane were all purchased from Fluka (Buchs, Switzerland). N-Biotinylcysteamine and Ncarboxyfluorescein (FAM)-cysteamine were prepared by reaction of cystamine with biotin succinimidyl ester and FAM-succinimidyl ester, respectively, FGESAGAAS and FGE(phosphoserine)AGAAS were prepared using standard Fmoc chemistry. FGES(O-isopropylmethylphosphonyl)AGAAS was prepared as described previously.5
Instrumentation. The mass spectrometer was a Q-TOF instrument (MicroMass, Altrincham, UK) equipped with a standard Z-spray electrospray interface. The LC system consisted of an Alliance 2690 HPLC gradient system (Waters, Milford, MA). Preparation of Procainamide Gel. The preparation of procainamide-Sepharose 4B gel was performed according to described procedures,9,10 with some slight modifications. A suspension of Sepharose 4B gel in ethanol/water (20%; 125 mL) was placed in a funnel with a glass filter and washed with water Analytical Chemistry, Vol. 78, No. 18, September 15, 2006
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Figure 3. Structure of the modified nonapeptide FGEXAGAAS, with X the converted phosphoserine residue, as analyzed after conversion of the phosphylated serine residue with 2-(3-aminopropylamino)ethanol by means of alkaline hydrolysis/Michael addition.
(1 L), resuspended in 0.2 M phosphate buffer (pH 11.5; 150 mL), and cooled at 4 °C. Cyanogen bromide (7 g in 15 mL of acetonitrile/water 1:1) was added gently, and the suspension was stirred for 10 min. Next, the suspension was washed with water (1 L) and immediately transferred to a solution of -aminohexanoic acid (1.6 g) in 0.2 M sodium carbonate, 0.2 M sodium bicarbonate, and 0.4 M sodium chloride, pH 9 (150 mL). The mixture was stirred for 48 h at 4 °C. Next, the gel was washed with water and resuspended in water (150 mL), and after addition of 4.2 g of procainamide hydrochloride, the pH of the mixture was adjusted to pH 4.5 with 1 M HCl. N-3-Dimethylaminopropyl-N-ethylcarbodiimide (7 g) was added, and the pH was kept at 4.5. When the pH remained relatively stable, the mixture was stirred at 4°C for 48-60 h. Next, the gel was washed with water (1.5 L). The wash fluid was collected, and the extinction was measured at 278 nm (278 ) 16 150 M-1 cm-1). Calculation revealed that 29 µmol of procainamide was bound to 1 mL of gel. The gel was stored in water (150 mL), containing 0.02% NaN3. Incubation of Plasma Samples with Organophosphates. Human plasma was incubated with sarin, soman, VX, or dichlorvos, to inhibit cholinesterase. The concentration of the OP in plasma was 3.7-7 µM, which is a 75-140-fold excess compared to the approximate concentration of HuBuChE in plasma (50 nM). Inhibition of the sample was allowed for 2 h at room temperature. As a blank, noninhibited plasma (0.5 mL) was used. The plasma samples were further processed as described below.
Isolation of HuBuChE from Human Plasma. A disposable 10-mL miniextraction column (tube ABIMED AMS 422 peptide synthesizer, Gilson, Villiers le Bel, France) was filled with 2 mL of procainamide-gel, which was washed with 20 mL of phosphate buffer (15 mM NaH2PO4 and 5 mM Na2HPO4, pH 6.9) Then, 1 mL of plasma sample was gently mixed with the procainamidegel. After 30 min at room temperature, the gel was washed with 5 mL of phosphate buffer and 5 mL of 150 mM sodium chloride (150 mM NaCl in phosphate buffer). Finally, HuBuChE was eluted with 7 mL of 600 mM NaCl in phosphate buffer. Digestion of HuBuChE with Pepsin. The HuBuChE solution obtained after procainamide affinity extraction was concentrated using a 100-kDa cutoff filter. The retentate was washed with 5% formic acid (2 × 2 mL). The retentate (∼200 µL) was transferred to a 4-mL glass vial; the filter was rinsed with 250 µL of 5% formic acid. The rinse fluid was combined with the retentate. Pepsin solution (50 µL of a 0.2% (i.e., 2 mg/mL) solution in 5% formic acid) was added. After incubation for 2 h at 37 °C, the incubation mixture was filtrated through a prewashed (0.5 mL of water) 3-kDa cutoff filter. The filter was washed with 150 µL of 5% formic acid solution, and the fluid was filtered and pooled with the first filtrate. This solution was used for LC-tandem MS experiments. LC-Tandem MS of Intact Pepsin Digests. Stationary phase was a PepMap C18 column (15 cm × 1 mm, 3-µm particles) from LC-Packings (Amsterdam, The Netherlands). The mobile phase consisted of a gradient of (A) 0.2% formic acid in water and (B) 0.2% formic acid in acetonitrile. Gradient program was 0-5 min, 100% A, flow 0.1 to 0.6 mL/min; 5-60 min, 100% A to 70% B, flow 0.6 mL/min. The pump flow (0.6 mL/min) was reduced to a column flow of ∼40 µL/min by a splitter (LC-Packings). Injection volume was 10 µL. Electrospray MS-MS spectra of the protonated molecular ion were recorded using a cone voltage of ∼35 V and a collision energy of ∼30 eV. Subsequently, ion chromatograms of m/z 778.4, the most selective fragment originating from the
Figure 4. Product ion mass spectrum of [M + 2H]2+, 448.7 Da, resulting from conversion of synthetic FGE(phosphoserine)AGAAS by alkaline hydrolysis and subsequent reaction with HOCH2CH2NHCH2CH2CH2NH2. 6642 Analytical Chemistry, Vol. 78, No. 18, September 15, 2006
Figure 5. Method for conversion of phosphylated HuBuChE into a common nonapeptide.
loss of the phosphyl moiety from the protonated molecular ion, were generated. Conversion of Pepsin Digests of HuBuChE with Ba(OH)2 and 2-(3-Aminopropylamino)ethanol and Subsequent LCTandem MS Analysis. Isolation of HuBuChE from plasma and subsequent pepsin digestion was carried out as described above. The filtrate was concentrated, coevaporated with 50 mM NH4HCO3 (2 × 0.5 mL), and dissolved in an aqueous solution of Ba(OH)2 and 2-(3-aminopropylamino)ethanol (100 and 50 mM, respectively; 0.2 mL). After incubation for 1 h at 37 °C, the reaction was quenched by the addition of acetic acid (10 µL). The resulting solution was analyzed with LC-MS/MS as described above, but with a collision energy of 18 eV. Safety Considerations. Sarin, soman, VX, and dichlorvos are highly toxic substances and should be handled with extreme care in fume cupboards. Skin or eye contact and accidental inhalation or ingestion should be avoided. RESULTS AND DISCUSSION We here describe a novel and generic assay for OP biomonitoring that is based on pepsin digestion of HuBuChE, followed by base-catalyzed β-elimination of the phosphyl moiety and subsequent Michael addition of a suitable nucleophile. This results in one common modified nonapeptide that can be analyzed by means of LC tandem MS. Initially, purified HuBuChE was completely inhibited with two different nerve agents (VX and soman) and subsequently sub(9) Ralston, J. S.; Main, A. R.; Kilpatrick, B. F.; Chasson, A. L. Biochem. J. 1983, 211, 243-250. (10) Grunwald, J.; Marcus, D.; Papier, Y.; Raveh, L.; Ashani, Y. J. Biochem. Biophys. Methods 1997, 34, 123-135.
jected to alkaline hydrolysis and subsequent Michael addition of ethanethiol. Subsequent digestion with pepsin resulted in both cases in FGE(S-ethylcysteine)AGAAS (see Figure 1 for an MS/ MS spectrum). Unfortunately, when we switched to plasma samples, it appeared that the alkaline hydrolysis of phosphylated HuBuChE and subsequent introduction of ethanethiol was troublesome, probably due to precipitation of various proteins in the alkaline matrix. We decided to perform the pepsin digestion of isolated HuBuChE prior to the combined alkaline treatment/Michael addition. Then, we performed experiments on spiked digests. To this end, pepsin digests of isolated HuBuChE (from blank plasma) were used for spiking experiments with synthetic FGE(O-isopropyl methylphosphonylserine)AGAAS (40, 80, and 160 ng/200 µL pepsin digest). After lyophilization and coevaporation with aqueous NH4HCO3 (50 mM; 2 × 250 µL)m a linear relationship was observed between amount of phosphopeptide and amount of converted nonapeptide, as determined with MS. We found that alkaline hydrolysis (100 mM Ba(OH)2/50 mM EtSH) for 1 h at 37 °C was long enough to convert the phosphoserine group almost quantitatively to an S-ethylcysteine group. It has been reported that unmodified serine hydroxyl groups are also susceptible to β-elimination under alkaline conditions,11 although to a much lesser extent. When these conditions Ba(OH)2/ethanethiol (100 and 50 mM, respectively) were applied to pepsin digests of isolated blank HuBuChE samples, FGE(S-ethylcysteine)AGAAS could not be detected. The drawback of using ethanethiol, unfortunately, was that the derived S-ethyl nonapeptide elutes in a rather “crowded” region of the chromatogram. Furthermore, the degree of fragmentation (11) Li, W.; Backlund, P. S.; Boykins, R. A.; Wang, G.; Chen, H.-C. Anal. Biochem. 2003, 323, 94-102.
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Figure 6. Ion chromatograms of m/z 791.4 in processed pepsin digests of isolated HuBuChE from plasma samples. Panel A represents the ion chromatogram of a processed digest from blank plasma. Panels B-E represent the ion chromatograms of digests from plasma that had been exposed to sarin, soman, VX, and dichlorvos, respectively. The digests were subjected to modification with 2-(3-aminopropylamino)ethanol, under the agency of Ba(OH)2. The arrows indicate the peak of the modified FGEXAGAAS. In the first MS, [M + 2H]2+, 448.7 was selected.
of the particular S-ethyl nonapeptide is very high (see Figure 1). To end up with more suitable nonapeptides, easily available synthetic FGE(phosphoserine)AGAAS was used advantageously to evaluate the use of nucleophiles other than ethanethiol; see Figure 2 for a selection of nucleophiles that were used. Evaluation criteria were based on both chromatography (matrix interference) and degree of fragmentation of the resulting nonapeptide. It appeared that the nonapeptide that resulted from reaction with 2-(3-aminopropylamino)ethanol (H) (see Figure 3 for the chemical structure) has both favorable chromatographic and mass spectrometric properties; a nice product ion mass spectrum of [M + 2H]2+ could be observed (see Figure 4). The eventual method (see Figure 5) proved to be viable with plasma samples that had been exposed to various OPs (see Figure 6). Two peaks were observed, which can be explained by the fact that due to the formation of a dehydroalanine residue and subsequent attack of the nucleophile on the double bond, the resulting amino acid loses its chiral integrity. Since the chirality of the other amino acids in the peptide does not change, a diastereomeric mixture of two peptides results. Performing the modification reaction on the peptide level has the additional advantage that a two-step approach can be followed. First, the generic method can be used for an initial screening of samples, and after finding a positive sample, the original pepsin digest can 6644 Analytical Chemistry, Vol. 78, No. 18, September 15, 2006
be analyzed in a more specific way as described by Fidder et al.,5 in order to unravel the identity of the OP inhibitor. It can be envisaged that the present method can be used as a rough screening method in case of large numbers of samples, as is to be expected after a terrorist incident. Subsequently, after finding a positive sample, the digest can be analyzed in a more specific way, by reanalyzing the original digest for phosphylated nonapeptides in a more thorough and laborious way. Furthermore, the method might be valuable for epidemiological studies for determination of total OP pesticide exposure. In future experiments, we will use a triple-quadrupole instrument in order to detect lower inhibition levels. In conclusion, with the presented method, HuBuChE inhibition can now accurately be monitored by mass spectrometry, without advance knowledge of the structure of the inhibitor. ACKNOWLEDGMENT The Centers for Disease Control and Prevention (CDC, Atlanta, GA) and the Dutch Ministry of Defense are gratefully acknowledged for financial support. Received for review May 24, 2006. Accepted July 13, 2006. AC060954T
