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Apr 24, 2014 - ABSTRACT: Shiga-like toxins (verotoxins) are a class of AB5 holotoxins that are primarily responsible for the virulence associated with...
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Safe and Effective Means of Detecting and Quantitating Shiga-Like Toxins in Attomole Amounts Christopher J. Silva,* Melissa L. Erickson-Beltran, Craig B. Skinner, Irina Dynin, Colleen Hui, Stephanie A. Patfield, John Mark Carter, and Xiaohua He Western Regional Research Center, United States Department of Agriculture, Albany, California 94710, United States S Supporting Information *

ABSTRACT: Shiga-like toxins (verotoxins) are a class of AB5 holotoxins that are primarily responsible for the virulence associated with Shiga-like toxin producing Escherichia coli (STEC) infections. The holotoxins are composed of a pentamer of identical subunits (B subunit) responsible for delivering the catalytic subunit (A subunit) to a host cell and facilitating endocytosis of the toxin into the cell. The B subunits are not associated with toxicity. We developed a multiple reaction monitoring method based on analyzing conserved peptides, derived from the tryptic digestion of the B subunits. Stableisotope-labeled analogues were prepared and used as internal standards to identify and quantify these characteristic peptides. We were able to detect and quantify Shiga toxins (Stx), Shigalike toxin type 1 (Stx1) and type 2 (Stx2) subtypes, and to distinguish among most of the known subtypes. The limit of detection for digested pure standards was in the low attomole range/injection (∼10 attomoles), which corresponded to a concentration of 1.7 femtomol/mL. A matrix effect was observed when dilute samples were digested in the buffer, Luria broth, or mouse plasma (LOD ∼ 30 attomol/injection = 5 femtomol/ mL). In addition, we determined that the procedures necessary to perform our mass spectrometry-based analysis completely inactivate the toxins present in the sample. This is a safe and effective method of detecting and quantitating Stx, Stx1, and Stx2, since it does not require the use of intact toxins.

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damage, by the addition of DNA-damaging antibiotics for example, then the lambdoid phages may reproduce lytically, by usurping control of the host’s metabolism.11 During lytic replication, intact phages are produced, stx genes are expressed and toxin molecules accumulate in the periplasmic space of the cell. Toxins and phages are released into the environment upon lysis of the bacteria by the phage, whereupon the process of phage infection, Stx production, and toxin release can begin anew. The Shiga-like toxins belong to the AB5 class of bacterial toxins. These toxins consist of five identical B subunits and a single catalytic A subunit.12,13 The five B subunits of Stx form a pentamer that binds to a host’s cell-surface ganglioside (globotriaosylceramide [Gb3Cer] or globotetraosylceramide [Gb4Cer])14−16 and delivers the A subunit, which, after internalization, is cleaved by the enzyme furin to yield the catalytically active A1 fragment, which is released from the A2 fragment upon reduction of a bridging cystine.17 The A1 subunit then translocates to the cytoplasm of the target cell, where it inactivates ribosomes with rRNA N-glycosidase

acterial food poisoning by Shiga-like toxin producing Escherichia coli (STEC) is a considerable worldwide health concern. The clinical manifestations of STEC infections can range from diarrhea to hemorrhagic colitis (HC) and potentially deadly hemolytic uremic syndrome (HUS).1−3 The STEC bacteria first adhere to intestinal epithelial cells and at a later time release their Shiga-like toxins. Shiga-like toxins (Stx, or verotoxins) are among the most important virulence factors in STEC infections. STEC produce two types of Shiga-like toxins: Stx1 and Stx2.4 Stx1 has a nearly identical amino acid sequence as Shiga toxin (Stx) from Shigella dysenteriae.4 Stx2 differs significantly from Stx1 but is a much more potent toxin in vivo.5−7 There are several subtypes of Stx within each type (Stx1: four subtypes; Stx2: seven subtypes), which vary in sequence, toxicity, and cellular targets. Stx are produced by genes under the control of lambdoid phages (Φ24B) that infect a variety of E. coli strains.8 The same E. coli bacterium may be infected by multiple lambdoid phages (Φ24B).9 The stx genes are under the control of late-phase phage promoters, such that when the phage is reproducing lysogenically (passively reproduced with the host genome), toxins are not normally produced or released. The expression of stx1 genes is additionally driven by iron-responsive bacterial promoters.10 If the phage-infected E. coli host suffers DNA © 2014 American Chemical Society

Received: September 13, 2013 Accepted: April 24, 2014 Published: April 24, 2014 4698

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Victor 3 plate reader (PerkinElmer, Waltham, Massachusetts). All treatments were performed in triplicate. Cells grown in a medium with added PBS (no toxin) were used as a negative control (100% viability = 0% toxicity). Cells grown in a medium containing 10 ng of Stx2a toxin were used as a positive control (0% viability = 100% toxicity). The percent cytotoxicity for cells was calculated as follows: [(cps from samples treated − cps from positive control)/(cps from negative control − cps positive control)] × 100. A dilution-response curve is summarized in Figure S-2 of the Supporting Information. Generation of Shiga-Like Toxin Samples. The strains of Shiga-like toxin producers are listed in Table S-3 (Supporting Information). A single colony of each strain was used to inoculate a separate 20 mL culture of Luria Broth (LB; Fisher Biosciences). The inoculated culture was grown overnight at 37 °C on an orbital shaker (250 rpm). The following day 10 mL of the overnight culture was used to inoculate 500 mL of LB supplemented with 50 ng/mL of mitomycin C (Sigma-Aldrich, Milwaukee, WI). The freshly inoculated culture was grown at 37 °C for 24 h on an orbital shaker (250 rpm). The resulting culture was centrifuged for 15 min at 5000g to pellet the cells. The cellular pellet was autoclaved, bleached, and discarded. The sterile filtered supernatants were analyzed by mass spectrometry (vide infra). Stx1, Stx2a, Stx2c, Stx2e (E167Q mutant), and Stx2g were purified from the appropriate sterile-filtered supernatant or lysate by previously described methods.38 The purified toxins were analyzed by mass spectrometry (vide infra). The plasma samples were isolated from sheep, mouse, or hamster whole blood. The blood samples were drawn in EDTA (anticoagulant) by a commercial vendor or experimental animals, according to protocols approved by the location animal care and use committee. The samples were centrifuged at 2000g for 15 min to pellet the red and white blood cells. The plasma was isolated for further analysis by mass spectrometry (vide infra). Reduction, Alkylation, and Tryptic Cleavage of ShigaLike Toxins. Twenty microliters of each sample was reduced, alkylated, and trypsin-digested. The samples consisted of one of the following: 20 μL of sterile filtered bacterial supernatant; 10 μL of sterile LB broth, 10 μL of plasma, or 1 μg of purified toxin, diluted to a final volume of 20 μL with buffer A (25 mM ammonium bicarbonate, pH 8.0); or 0.1 μL of sterile filtered bacterial supernatant diluted into 10 μL of buffer A and spiked into 10 μL of buffer A, LB broth or plasma. Ten microliters of a freshly prepared solution of 10 mM dithiothreitol (DTT) in buffer A was added to the sample solution and allowed to react at 37 °C for 1 h and then cooled to room temperature. Forty microliters of a freshly prepared solution of buffer A containing 10 mM iodoacetamide was added to the solution and allowed to stand in the dark at room temperature for 1 h. The excess iodoacetamide was quenched by addition of 20 μL of a solution containing 10 mM DTT in buffer A. The reduced and alkylated peptides were subjected to proteolysis by the addition of 10 μL of a trypsin solution (100 μg of trypsin/mL of water). The reaction was allowed to proceed at 37 °C for 16 h. After the completion of the trypsin digestion, samples were filtered through a 10000 MWCO filter (12 min; 14000g). Samples were stored at −20 °C until they were analyzed. Mass Spectrometry. The instrument response was optimized by a previously described method.39 The qualitative mass spectrometry was performed using an Applied Biosystems (ABI/MDS SCIEX, Toronto, Canada) model QStar Pulsar

activity. It was once thought that Stx derived most of its toxicity from its N-glycosidase activity, but now other cellular processes (such as apoptosis) have been recognized to play a role in its pathogenicity.18 Shiga-like toxins have been detected using a number of different methods. The most common means of detection is to perform PCR on an isolate to determine whether it contains the toxin gene. This approach is limited, since the production of the toxin is regulated by the phage, so the presence of the toxin gene does not guarantee the expression of that toxin. Several antibody-based methods of detecting toxins have been developed, but they are only available for some subtypes.19−21 Mass spectrometry has been used to study the structure of the holotoxins22−32 but has not been used as a method of detection. Since the toxin is ultimately responsible for the observed symptoms, development of an assay that detects all of the Shiga and Shiga-like toxins would be extremely valuable. The multiple reaction monitoring (MRM) method is a wellestablished method of detecting and quantitating proteins that does not require the intact protein for analysis.33−35 A purified sample of the analyte protein is first reduced and alkylated to cleave and prevent the reformation of any disulfide bonds. The reduced and alkylated protein is then digested with trypsin (or other protease) to yield a set of peptides that are characteristic of the analyte protein. The resulting set of peptides is qualitatively analyzed by mass spectrometry to empirically identify peptides which have chromatographic properties (e.g. peak shape and retention time) and signal intensities appropriate for a MRM analysis. The instrument parameters for those peptides deemed suitable for MRM analysis are optimized separately for each peptide. The optimized peptide producing the most intense signal is selected as the analyte peptide, and the others may be used as confirmatory peptides. This approach typically allows the detection and quantification of peptides in the attomole (10−18 mole) range. In this way, the MRM method allows for the detection and quantification of a protein without analyzing the intact protein. This approach is especially appealing from a safety standpoint when analyzing toxins. We wish to report our MRM-based method of detecting and discriminating among the known Shiga and Shiga-like toxins, which is safe and effective in the attomole range, making it among the most sensitive Stx detection methods.

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EXPERIMENTAL PROCEDURES Measurement of Inactivation of Stx2a by Cytotoxicity to Vero Cells. An in vitro cytotoxicity assay was used to evaluate the inactivation of the Stx2a toxin (Verotoxin) using the well-established Vero cell-based assay.36,37 Fresh Vero cells were dispensed on 96-well plates at 1 × 105 cells/mL (100 μL/ well) and grown overnight in Dulbecco’s Modified Eagle Medium (DMEM, Invitrogen, Carlesbad, Calfornia) supplemented with 10% fetal calf serum (Invitrogen) and 1× Glutamax (Invitrogen) in a humidified incubator (37 °C, 5% CO2). Cells were first treated with samples (treated and untreated Stx2a dilutions and controls) diluted in fresh medium and incubated at 4 °C for 1 h. After incubation, the medium containing the sample was removed and replaced with fresh medium. The cells were incubated overnight at 37 °C in the fresh medium. The cytotoxicity was assessed using CellTiterGlo reagent (Promega, Fitchburg, Wisconsin), according to the manufacturer’s instructions, except that the reagent was diluted 1:5 in PBS prior to use. Luminescence was measured with a 4699

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Safety Considerations. Acetonitrile is hazardous and was manipulated in a dedicated chemical safety hood. Shiga-like toxins are dangerous substances and must be handled in a dedicated biosafety level 2 (BSL-2) laboratory using procedures outlined in the fifth edition of the CDC’s biosafety manual, Biosafety in Microbiological and Biomedical Laboratories (www.cdc.gov/biosafety/publications/bmbl5). The toxins were inactivated by a combination of reduction/alkylation/ trypsin digestion followed by filtration through a 10000 MWCO filter. The resulting filtrate, containing peptides and no active toxin, was subjected to our mass spectrometry-based analysis.

instrument equipped with a nanoelectrospray source. An Applied Biosystems (ABI/MDS Sciex, Toronto, ON) model 4000 Q-Trap instrument equipped with a nanoelectrospray source was used for quantification. This mass spectrometer was operated in multiple reaction monitoring (MRM) mode, alternating between detection of the nine peptides and the corresponding 15N-labeled internal standards. The mass settings for the peptides are summarized in Tables S-1 and S2 of the Supporting Information. Quantification was done with the IntelliQuant quantification algorithm using Analyst 1.5. Nanospray LC−MS/MS. An Applied Biosystems (ABI/ MDS Sciex, Toronto, ON) model 4000 Q-Trap instrument equipped with a nanoelectrospray source was used to perform nanospray liquid chromatography and tandem mass spectroscopy (LC−MS/MS). An aliquot (6 μL) of each digest was loaded onto a C-18 trap cartridge [Acclaim PepMap100, 5 μm, 100 Å, 300 μm (inside diameter) × 5 mm (Dionex, Sunnyvale, CA)]. Salts were washed from the cartridge with an acetic acid/ acetonitrile/heptafluorobutyric acid/water solution (0.5/1/ 0.02/99). The now salt-free bound peptides were eluted onto a reversed-phase column [Vydac (Hesperia, CA) 238EV5.07515, 75 μm × 150 mm]. The solvents were delivered with an Applied Biosystems model Tempo nanoflow LC system (ABI/MDS Sciex) with an autosampler, a column switching device, and a nanoflow solvent delivery system. Samples were eluted from the column with a binary gradient (A, 0.5% acetic acid in water, and B, 80% acetonitrile with 0.5% acetic acid). The flow rate was 250 nL/min with a 16 min linear gradient starting with 5% B and ending with 100% B. Elution with 100% B was conducted for 7 min followed by a return to 5% B over 4 min. The eluted samples were sprayed with a noncoated spray tip (FS360-20-10-N-20-C12, New Objective Inc., Woburn, MA) onto the Applied Biosystems source, model Nanospray II. Peptides, Internal Standards and Purified Proteins. The natural abundance (14N) peptides YNEDDTFTVK, YNENDTFTVK, YNGDNTFTVK, YNEDNTFTVK, IEFSK, EYWTSR, EYWTNR, ELFTNR, and VEYTK were obtained from Peptide 2.0 (Chantilly, VA). These peptides were at least 95% pure, and their structure was verified by mass spectrometry. The peptides were dissolved in 200 μL of water/acetonitrile buffer and the absorbance at 280 nm was recorded for these solutions. The absorbance was used, in conjunction with a calculated extinction coefficient (ExPASy; www.expasy.org/tools/water), to determine the peptide concentration. The cloning of the B subunits from Stx1 and Stx2a, Stx2b, Stx2c, Stx2d, Stx2e, Stx2f, and Stx2g was performed using standard molecular biology techniques.40 The 15N-labeled internal standards were prepared by growing the clones in minimal medium supplemented with 15NH4Cl (99.7% 15N; Cambridge Isotope, Andover, MA) and purified using standard molecular biology techniques (Supporting Information).41 The purity of the 15N-labeled B subunits was greater than 90% by Coomassie-stained SDS−PAGE gel. The B subunits were quantified according to the manufacturer’s instructions by using a commercially available BCA Protein Assay Kit (Pierce, Rockford, IL). The Shiga-like toxins were isolated by previously reported methods.42,43 The purity was judged to be greater than 90% by Coomassie-stained SDS−PAGE gel. The proteins were quantified by using a commercially available BCA Protein Assay Kit (Pierce, Rockford, IL) (Supporting Information).

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RESULTS AND DISCUSSION Using a Vero Cell-Based Assay to Ensure Tryptic Stx Treatments Were Nontoxic before Performing LC−MS/ MS Analysis. Vero cells were used to assay the cytotoxicity of Stx2a at the various steps of preparation. These cells have been shown to produce the cell-surface ganglioside necessary for Shiga-like toxin binding, which makes them extremely sensitive to Shiga-like toxins (verotoxins). Serial dilutions of the Stx2a toxin were performed to determine the Stx2a concentrations at 50% cell death (CD50) and the limit of detection by this assay.6,44−47 The percent cell viability was calculated relative to the PBS control and graphed (Figure S-2 of the Supporting Information). The limit of detection for the assay was determined to be between 1 and 10 picograms (∼13−130 attomoles) of toxin per well. Neither addition of trypsin nor the PBS control had any discernible effect on the cells. These results indicated that the assay had a 10000-fold dynamic range (1 pg to 10 ng/well). The effects of the reagents used in our mass spectrometrybased analysis on the Vero cells were examined. PBS and 10 ng of Stx2a toxin in PBS were used as the negative and positive controls, respectively (Figure 1, panels A and B). The reagent

Figure 1. Summary of Vero cell assay of the treated samples. Scale: 100% cell viability = no toxicity; 0% viability = 100% toxicity. (A) PBS alone. (B) Ten nanograms of Stx2a in PBS. (C) PBS with 3 mM DTT. (D) PBS with 10 mM IA. (E) PBS sequentially treated with DTT, IA, DTT. (F) PBS sequentially treated with DTT, IA, DTT, and then digested with trypsin. (G) PBS sequentially treated with DTT, IA, DTT, digested with trypsin, and then filtered through a 10000 MWCO filter. (H) Ten nanograms of Stx2a treated with 3 mM DTT. (I) Ten nanograms of Stx2a incubated overnight with trypsin. (J) Ten nanograms of Stx2a sequentially treated with DTT, IA, DTT. (K) Ten nanograms of Stx2a sequentially treated with DTT, IA, DTT, and then digested with trypsin. (L) Ten nanograms of Stx2a sequentially treated with DTT, IA, DTT, digested with trypsin, and then filtered through a 10000 MWCO filter. (M) Ten nanograms of Stx2a filtered through a 10000 MWCO filter. 4700

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were compiled and compared (Figure S-3 of the Supporting Information). On the basis of this analysis, two additional peptides, YNGDNTFTVK (Stx2g) and YNEDNTFTVK (Stx2e), in combination with the other peptides, would be sufficient to detect all known Stx2 subtypes. These seven peptides were empirically deemed most suitable to be analyte peptides for a multiple reaction monitoring (MRM)-based method of analysis. MRM Optimization of the Seven Stx2 Tryptic Peptides. The seven tryptic peptides were commercially synthesized and obtained in at least 95% purity. The source parameters and the Q2 offset voltage (“collision energy”) were adjusted separately for each peptide to yield optimal fragmentation (Table S-1 and S-2 of the Supporting Information). The optimal fragments of the IEFSK peptide were the y3 and y4 ions (containing the amino acids FSK and EFSK, respectively).48 The peptides EYWTSR and EYWTNR fragment similarly, and the optimal fragments were the y3 and y4 ions (corresponding to amino acids TSR and WTSR and TNR and WTNR, respectively). The other four peptides (YNEDDTFTVK, YNENDTFTVK, YNGDNTFTVK, or YNEDNTFTVK) were observed to fragment in very similar fashions, and in all cases, the y8 fragment ion (containing amino acids EDDTFTVK, ENDTFTVK, GDNTFTVK, or EDNTFTVK, respectively) produced the most intense signals. In addition, these conditions were determined to be optimal for the b2, y7, and y6 ions of all four peptides. The relative intensity of the y8, b2 (containing amino acids YN), y7 (containing amino acids DDTFTVK, NDTFTVK, or DNTFTVK, respectively), and y6 (containing amino acids DTFTVK or NTFTVK, respectively) ions were determined to be 1.0, 0.7, 0.5, and 0.2, respectively, for peptides YNEDDTFTVK, YNENDTFTVK, and YNEDNTFTVK. The relative intensities for for y8, b2, y7, and y6 ions for the peptide YNGDNTFTVK were determined to be 1.0, 0.5, 0.1, and 0.1 respectively. These optimized fragmentations were used to detect and discriminate among the analyte peptides in subsequent experiments. MRM Optimization of the Two Stx1 Tryptic Peptides. A Stx1 tryptic peptide (ELFTNR) was commercially synthesized (>95% purity) and another obtained from the trypsin digestion of Stx1 toxin (YNDDDTFTVK). The source parameters and the Q2 offset voltage (“collision energy”) were adjusted for each peptide to yield optimal fragmentation (Tables S-1 and S-2 of the Supporting Information). The y8 fragment ion produced the most intense signal for the YNDDDTFTVK. The relative intensities for y8, b2, y7, and y6 ions for this peptide were determined to be 1.0, 0.6, 0.3, and 0.2, respectively. The optimal peptide fragments from the ELFTNR were determined to be the y3 and y4 ions. Additionally, the chromatographic properties and molecular weight differences for these two peptides were sufficient to permit us to distinguish among the Stx1 (or Stx) and Stx2 subtypes. Selection of the Analyte Peptide. Samples of purified Shiga toxins Stx1 and Stx2a were digested with trypsin and analyzed by mass spectrometry. The relative signal intensities of the y4 ions from IEFSK or EYWTSR and the y8 ion from YNEDDTFTVK from Stx2a were compared and determined to be 0.5:1:1, respectively. The relative signal intensities of the y4 ion from ELFTNR and the y8 ion from YNDDDTFTVK from Stx1 were determined to be 1:1. At higher dilutions (∼1 fmol/ injection), the ratio of the signal intensity of the y4 ion of EYWTSR to the y8 ion from YNEDDTFTVK was reduced to

diothiothreitol (DTT) was shown to have no deleterious effects on cell growth (Figure 1C), although iodoacetamide (IA) alone showed deleterious effects (Figure 1D). A mock reduction/ alkylation procedure was performed by incubating DTT with just buffer (without toxin), then IA was added, and, after a 1 h incubation, an excess of DTT was added to quench any excess IA (DTT-IA-DTT). This mock reaction mixture was then digested with trypsin (DTT-IA-DTT + tryspin) and was filtered after the overnight digestion (DTT-IA-DTT + tryspin + filtration). Aliquots of these three steps were analyzed and shown to have no deleterious effect on the cells (Figure 1, panels E, F, and G). This demonstrated the reduction/ alkylation sequence (DTT-IA-DTT), the trypsin digestion (DTT-IA-DTT + tryspin), and subsequent filtration (DTT-IADTT + tryspin + filtration) sequence had no deleterious effects on the Vero cell growth. Using Vero Cells to Assay the Inactivation of Stx2a Toxin during Sample Preparation Prior to LC−MS/MS Analysis. Ten nanograms of Stx2a toxin was used in each assay. This amount was shown to be approximately 100 times more than the minimum amount necessary to kill all of the cells in the well (Figure S-2 of the Supporting Information). The Stx2a toxin was not inactivated by treatment with DTT alone or by overnight digestion with trypsin alone (Figure 1, panels H and I, respectively). Reduction/alkylation (DTT-IA-DTT) of the toxin was sufficient to completely inactivate it (Figure 1J). Subsequent digestion with trypsin (DTT-IA-DTT + tryspin) (Figure 1K) and filtration (DTT-IA-DTT + tryspin + filtration) (Figure 1L) were also shown to completely inactivate the toxin. Even simple filtration of the intact toxin through a 10000 MWCO filter was demonstrated to remove the toxin (Figure 1M). These results indicate that the toxin was inactivated (>10000-fold) by the steps necessary to prepare it for mass spectrometry-based analysis. Shiga-like toxins are extremely toxic substances. The LD50 for mice is estimated to be 50 ng/kg (∼0.7 pmol/kg).6,7 Although mass spectrometry requires only a miniscule amount of toxin for analysis, there is always concern that electrospraying may deposit some of the toxin on the instrument, where it could accumulate. Hypothetically, if the toxins are not inactivated, they could accumulate on the instrument to eventually achieve a sufficient concentration to be a health concern. It is also possible that the amounts involved may represent a health hazard from inhalation. However, based on our results, the initial reduction/alkylation/reduction procedure is sufficient to irreversibly inactivate the toxin. The subsequent trypsin cleavage then converts the inactive proteins into peptides. The filter retains any intact proteins and allows the tryptic peptides to pass through. Thus, our approach ensures that the samples are fully inactivated prior to analysis. Furthermore, since the proteins are digested to peptides, they cannot be reconstituted. Qualitative LC−MS/MS Analysis of Tryptic Peptides from Purified Stx2a and Stx2b Toxins. Stx2a and Stx2b were separately digested with trypsin, and the resulting sets of tryptic peptides were subjected to a qualitative LC−MS/MS analysis. The peptides YNEDDTFTVK (Stx2a), YNENDTFTVK (Stx2b), IEFSK (Stx2a and Stx2b), EYWTSR (Stx2a), and EYWNTR (Stx2b) from the B subunits of the toxins were found to be suitable for further MRM optimization. Each produced an intense signal and each peptide also had very good chromatographic properties (e.g., peak shape and retention time). The known sequences of the B subunits of Stx2 subtypes 4701

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Five femtomoles of Stx2a was spiked into the buffer, Luria broth (LB), or mouse plasma and then subjected to the reduction/alkylation/trypsin digestion to yield a final volume of 1 mL. These samples were injected multiple times (n = 10) (Figure S-7 of the Supporting Information). The signals from these samples corresponded to ∼30 attomoles per injection or 5 femtomol/mL. These signals were of lower intensity than those observed from the analysis of the diluted digests of Stx2a (vide supra), which indicated a matrix effect. The signal may be improved by affinity concentrating samples from the matrix before analysis, using available methods.42,49 The LOD value of 10 attomoles for a dilution of a tryptic digestion of purified toxin corresponds to an absolute amount. Typical assays for Shiga-like toxins report values as concentrations. On the basis of a 6 μL injection, this LOD amounts to a concentration of approximately 1.7 fmol/mL for diluted digests of pure toxins. When 5 femtomoles are digested in buffer, LB, or mouse plasma, the LOD corresponds to approximately 5 fmol/mL (30 attomol/injection). This value is comparable to the LD50 for an animal bioassay (14 fmol/20 g mouse). In addition, if a sample is concentrated or affinity purified by available methods42,49 then the amount of toxin detectable in a given volume can be reduced further up to the LOD of the instrument (10 attomole/injection). Using the Physicochemical Properties of the Tryptic Peptides to Distinguish Among the Stx1 Toxin and Stx2 Toxin Subtypes. The peptide YNGDNTFTVK (Stx2g) was readily distinguished from the other three by virtue of having a precursor ion that is very different (579.0) from the others (615.8 and 616.3) and a different chromatographic retention time. YNEDDTFTVK (Stx2a and Stx2f) was distinguished from YNENDTFTVK (Stx2b, Stx2c, and Stx2d) and YNEDNTFTVK (Stx2e), by chromatographic retention times, even though the peptides had a similar precursor (616.3 vs 615.8) and y8 product ions (954.4 vs 953.4). The peptides YNEDNTFTVK (Stx2e) and YNENDTFTVK (Stx2b, Stx2c, and Stx2d) were shown to have identical amino acid compositions, precursor ions, and had very similar chromatographic properties. If the signal from the y6 ion was examined, the y6 ion signal for the peptide YNENDTFTVK (Stx2b, Stx2c, and Stx2d) was intense, while the analogous signal for transition 615.8 to 709.4 was indistinguishable from the instrument background. In contrast, when the same transitions were examined for the peptide YNEDNTFTVK (Stx2e), a strong signal was observed for the 615.8 to 709.4 transition and a noticeably less intense signal was seen for the 615.8 to 710.4 transition, due to the contribution of natural abundance 13C atoms (∼1.1% of the carbon atoms) present in the sample (Figure S-8 of the Supporting Information). In this way, all four analyte peptides could be distinguished. The peptides EYWTSR and EYWTNR were shown to be readily distinguishable due the differences in their molecular weights. Stx2a and Stx2f were observed to share a common analyte peptide (YNEDDTFTVK). A similar observation was made for Stx2b, Stx2c, and Stx2d (YNENDTFTVK). Trypsin digestion of Stx2a, Stx2c, or Stx2d yielded the peptide EYWTSR, while the peptide EYWTNR was produced from a digestion of Stx2b, Stx2e, Stx2f, or Stx2g. These results indicated that the EYWTSR and EYWTNR peptides could be used to distinguish among some of the Stx2 subtypes that shared a common analyte peptide. By including the analyte peptides, EYWTSR and EYWTNR, all of the Stx2 toxins could

0.8. The interference from the chemical noise was also determined to be less for the higher molecular weight peptides, than the lower molecular weight ones (data not shown). For these reasons, the peptides YNEDDTFTVK, YNENDTFTVK, YNEDNTFTVK, YNGDNTFTVK, and YNDDDTFTVK were selected to be the analyte peptides. Limit of Detection for the Analyte Peptides. Serial dilutions of the synthetic analyte peptides were prepared and analyzed using this MRM method. The graphs of the signal intensities for the y8 ion for all four peptides are summarized in Figure 2. The signal intensities for the 25 attomole samples are

Figure 2. LOD for the four analyte peptides (synthetic). Graphs show the signal intensity of the y8 ion of the peptides (A) YNEDDTFTVK, (B) YNENDTFTVK, (C) YNGDNTFTVK, and (D) YNEDNTFTVK from either a (i) 200, (ii) 100, (iii) 50, (iv) 25, or (v) 0 attomole injection. The graphs are offset for clarity.

similar for all four of the peptides. This indicates that the LOD is between 25 and 50 attomoles (S/N > 3 × background). Since there are five identical B subunits per toxin molecule, the LOD for the toxin is in the low attomole range (∼10 amol/injection), based on the use of pure standards. One microgram samples of purified Stx1 and Stx2a toxin were digested with trypsin and diluted and analyzed by mass spectrometry. The Stx2a sample was diluted 1000, 10000, and 100000 times before analysis. These dilutions corresponded to approximately 800, 80, or 8 attomol/injection (Figure S-4 of the Supporting Information). A similar limit of detection was also observed when 8 attomoles or 16 attomoles of a tryptic digestion of Stx1a or Stx2a was injected multiple times (n = 20) (Figures S-5 and S-6 of the Supporting Information). These results indicate that the LOD for the analyte peptide derived from a synthetic peptide or from the trypsin digest of a purified toxin is approximately 10 attomoles. 4702

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Figure 3. Detection and quantification of toxin subtypes present in LB supernatants. Graphs show the relative signal intensity of the (i) y8 ion from Shiga-like toxins and the (ii) y8 ion from the corresponding 15N-labeled internal standard. The Shiga-like toxins were detected in the LB supernatants used to grow eight different strains of STEC. (A) Stx2a, (B) Stx2b, (C) Stx2c, (D) Stx2d, (E) Stx2e, (F) Stx2f, (G) Stx2g, and (H) Stx1a. The internal standard was added to the trypsin digestion mixture. The signal intensities of the (ii) y8 ions derived from 15N-labeled internal standards are normalized to 500. The signal intensities of the y8 ions from the toxins are not normalized. The retention times are reported as relative retention times. The signals for (A) Stx2a, (C) Stx2c, and (D) Stx2d correspond to 12 nanoliters of supernatant. The signals for (E) Stx1e and (H) Stx1a correspond to 120 nanoliters of medium per injection. The signals for (B) Stx2b, (F) Stx2f, and (G) Stx2g correspond to 400 nanoliters of medium per injection. The signal intensities correspond to 6 × 101 ± 1 × 101 amoles of (A) Stx2a, (B) 2.3 × 102 ± 3 × 101 amoles of Stx2b, (C) 1.9 × 102 ± 2 × 101 amoles of Stx2c, (D) 1.2 × 102 ± 1 × 101 amoles of Stx2d, (E) 2 × 101 ± 2 amoles of Stx2e, (F) 3.0 × 102 ± 1 × 101 amoles of Stx2f, (G) 9.6 × 102 ± 1 × 101 amoles Stx2g, and (H) 1.6 × 103 ± 2 × 102 amoles Stx1a.

be detected, and it was possible to distinguish among six (Stx2c and Stx2d are indistinguishable) of the seven Stx2 subtypes. Since the production of toxins is governed by phages, it is anticipated that new strains of STEC will emerge. If a phage from one STEC infects a new strain of E. coli, then it may become a new strain of STEC.50−52 The direct detection, quantification, and identification of the toxin molecule or molecules is/are necessary, since the presence of the toxin is the greatest determinant of virulence, whereas the presence of a toxin gene is not a guarantee that the toxin is produced. Even if the toxin gene is present and the toxin is produced, it may be produced in dramatically varying amounts, as shown in Figures 3 and 4. Direct detection of the toxin is crucial for identifying the source of the toxin. The parameters used to detect the analyte peptide for Stx1 (YNDDDTFTVK) are similar to those used to detect the analyte peptides of the Stx2 subtypes. Trypsin digestion of other subtypes of the Stx1 would yield homologous peptides, YNDDDSFTVK and YNDDDTFTAK.53 By adjusting the method to incorporate the appropriate transitions for these molecules, this method can readily be adapted to include these compounds. These changes will permit a researcher to detect any subtype of Stx, be it Stx1, Stx2, or Stx from the Shigella genus. As other subtypes are discovered, this method can rapidly be adapted to include them as well. The versatility of

this method is highlighted by its ability to detect both Stx1 and Stx2 within a single sample or from a single isolate (Figure 4). Although separate detection of Stx1 and Stx2 is routine using immunological techniques, it is much more difficult to distinguish between two closely related subtypes in the same mixture (such as Stx2a and Stx2c, which are frequently found together in the same STEC strain).54,55 This would be straightforward using this MRM method, and this may be the only method capable of discerning between Stx2a and Stx2c in the same sample. The appropriate isotopically labeled internal standards permit the detection, discrimination, and quantification of the Stx1 and Stx2 present in a sample. Preparing the 15N-Labeled Internal Standards. The B subunit was demonstrated to be essential for binding the toxin to the gangliosides present on the external cell membrane, but it was not toxic by itself. Primers were designed to amplify the gene sequences of the major subtype of Stx1 (Stx1a) and seven known B subunits of Stx2 (Stx2a, Stx2b, Stx2c, Stx2d, Stx2e, Stx2f, and Stx2g) (Table S-4 of the Supporting Information). The resulting clones were sequenced and shown to express the intact B subunit and a C-terminal His tag to facilitate purification by immobilized metal affinity chromatography (IMAC). The plasmids were cloned into BL21 cells, which were grown in minimal medium supplemented with 15NH4Cl. The resulting 15N-labeled His-tagged B subunits were purified 4703

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summary, the 15N-labeled proteins are of high isotopic purity and, upon trypsin digestion, yield no peptides that would interfere with this analysis. Assessing the Background Interference with the Analyte Peptides. The trypsin digestion of a toxin resulted in a large mixture of nonanalyte tryptic peptides, so it was necessary to determine if these other peptides would interfere with the analysis. Five purified toxins [Stx1, Stx2a, Stx2c, Stx2e (E167Q mutant), and Stx2g] were digested with trypsin and analyzed by mass spectrometry. This analysis indicated that the other peptides derived from the tryptic digestion of the Stx1 toxin did not interfere with the detection of the Stx2 subtypes. The converse was also true (Figures S-10 and S-11 of the Supporting Information). It was necessary to determine whether any molecules present in the complex medium (Luria broth) where STEC are grown to induce toxin production might interfere with this analysis. An interfering molecule would have to have an identical chromatographic retention time and identical precursor and product ions. The samples were spiked with the appropriate 15 N-labeled internal standards. Medium from lysed non-STEC E. coli O6 was analyzed by this method. Samples of sheep, mouse, and hamster plasma were subjected to a similar analysis. Upon examination of the resulting chromatograms, no interfering molecules were found (Figure S-12 of the Supporting Information). Calibration Curves for the Quantification of ShigaLike Toxins in a Sample. The amount of synthetic analyte peptide provided by the vendor was determined by measuring its absorbance at 280 nm. The 15N internal standards were obtained from the trypsin digest of the appropriate purified 15 N-labeled His-tagged B subunit. A set of solutions containing a fixed amount of the appropriate 15N-labeled internal standard and varying amounts of the corresponding synthetic peptide were prepared for each of the five analyte peptides. These solution sets were analyzed by mass spectrometry. The area ratio of the signal from the y8 ion of the synthetic peptide to the y8 ion of the analogous 15N-labeled internal standard was calculated (n = 4) for each solution. These data were used to prepare a calibration curve relating the area ratio of the signal from the y8 ion from a known amount of synthetic peptide to the y8 ion from a fixed amount of the corresponding isotopically labeled internal standard (Figures S-13 and S-14 of the Supporting Information). The curves were determined to be linear over a >100-fold range with an excellent correlation coefficient (>0.99). Quantifying Shiga-Like Toxins in a Sample. Clinical isolates of STEC producing Stx2a, Stx2b, Stx2c, Stx2d, Stx2e, Stx2f, Stx2g, or Stx1a and two strains producing both Stx1 and Stx2a were grown in LB and induced to produce Shiga-like toxin by the addition of the antibiotic mitomycin C (Table S-3 of the Supporting Information).43 An aliquot of the filtersterilized supernatants from each of these samples was digested with trypsin, and a fixed amount of the appropriate trypsin digested 15N-labled internal standard was added. The nine samples were analyzed by MS, and the area ratio of the signal from the y8 ion from the analyte and corresponding 15N-labeled peptide was calculated. The previously described calibration curves were used to quantify the amount of toxin present in the ten previously described samples. These data are listed in the legends of Figures 3 and 4. We were able to detect and quantify all of the Stx2 toxin subtypes in the attomole range [20 (Stx2e) to 960 (Stx2g) attomoles)] (Figure 3). We were also able to

Figure 4. Simultaneous detection and quantification of two toxin subtypes present in LB supernatants. Graphs show the relative signal intensity of the y8 ion of the analyte peptide from either (i) Stx1 or Stx2 or (ii) the corresponding 15N-labeled internal standard ions. Samples A and B are derived from the supernatant of two different strains of STEC grown in LB. Both strains simultaneously produce Stx1 and Stx2a toxins. The signal intensities for the y8 ions derived from (ii) 15N-labeled internal standards are normalized to 500. The signal intensities for the y8 ion from the analyte peptide derived from the Stx1 or Stx2 toxins are not normalized. The retentions times are reported as relative retention times. The signals correspond to 24 nanoliters of medium per injection. The signal intensities for sample A correspond to a toxin concentration of 1.0 × 103 ± 3 × 102 amoles of Stx1 and 2.0 × 103 ± 2 × 102 amoles of Stx2a. The signal intensities for sample B correspond to a toxin concentration of 4.0 × 102 ± 2 × 102 amoles of Stx1 and 7 × 101 ± 2 amoles of Stx2a.

by IMAC and used as stable-isotope internal standards. The purity of these proteins was estimated to be greater than 90% (Figure S-1 of the Supporting Information). The internal standards were derived from cloned proteins, so it needed to be determined if the other peptides produced by the trypsin digestion of these proteins would interfere with the analysis. Samples of four purified 15N-labeled His-tagged B subunit proteins from Stx1a, Stx2a, Stx2c, Stx2e, and Stx2g were analyzed by our mass spectrometry-based method. We determined that none of the tryptic peptides derived from the 15N-labeled proteins interfered with the analysis of the Stx, Stx1, or Stx2 toxins (Figures S-9 of the Supporting Information). Additionally, we injected approximately 100 fmol of the tryptic peptides from each of the five 15N-labeled internal standards and analyzed them by mass spectrometry. The resulting chromatograms were examined, and we saw no signal above noise for the corresponding natural abundance (14N) peptide (data not shown), which indicated that the 15Nlabeled internal standards were of very high isotopic purity. In 4704

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(5) Fuller, C. A.; Pellino, C. A.; Flagler, M. J.; Strasser, J. E.; Weiss, A. A. Infect. Immun. 2011, 79, 1329−1337. (6) Lindgren, S. W.; Samuel, J. E.; Schmitt, C. K.; O’Brien, A. D. Infect. Immun. 1994, 62, 623−631. (7) Tesh, V. L.; Burris, J. A.; Owens, J. W.; Gordon, V. M.; Wadolkowski, E. A.; O’Brien, A. D.; Samuel, J. E. Infect. Immun. 1993, 61, 3392−3402. (8) Tyler, J. S.; Livny, J.; Friedman, D. I. In Phages; Their Role in Pathogenesis and Biotechnology.; Waldor, M. K., Friedman, D. I., Adhya, S. L., Eds.; ASM Press: Washington, D. C., 2005; pp 131−164. (9) Smith, D. L.; Rooks, D. J.; Fogg, P. C.; Darby, A. C.; Thomson, N. R.; McCarthy, A. J.; Allison, H. E. BMC Genomics 2012, 13, 311. (10) Wagner, P. L.; Livny, J.; Neely, M. N.; Acheson, D. W.; Friedman, D. I.; Waldor, M. K. Mol. Microbiol. 2002, 44, 957−970. (11) Friedman, D. I.; Court, D. L. Curr. Opin. Microbiol. 2001, 4, 201−207. (12) Fraser, M. E.; Chernaia, M. M.; Kozlov, Y. V.; James, M. N. Nat. Struct. Biol. 1994, 1, 59−64. (13) Fraser, M. E.; Fujinaga, M.; Cherney, M. M.; Melton-Celsa, A. R.; Twiddy, E. M.; O’Brien, A. D.; James, M. N. J. Biol. Chem. 2004, 279, 27511−27517. (14) Ling, H.; Boodhoo, A.; Hazes, B.; Cummings, M. D.; Armstrong, G. D.; Brunton, J. L.; Read, R. J. Biochemistry 1998, 37, 1777−1788. (15) Sandvig, K. Toxicon 2001, 39, 1629−1635. (16) Shimizu, T.; Ohta, Y.; Noda, M. Infect. Immun. 2009, 77, 2813− 2823. (17) Garred, O.; van Deurs, B.; Sandvig, K. J. Biol. Chem. 1995, 270, 10817−10821. (18) Tesh, V. L. Curr. Top. Microbiol. Immunol. 2010, 357, 137−178. (19) He, X.; Qi, W.; Quinones, B.; McMahon, S.; Cooley, M.; Mandrell, R. E. Appl. Environ. Microbiol. 2011, 77, 3558−3564. (20) Feng, P. C.; Jinneman, K.; Scheutz, F.; Monday, S. R. Appl. Environ. Microbiol. 2011, 77, 6699−6702. (21) Willford, J.; Mills, K.; Goodridge, L. D. J. Food Prot. 2009, 72, 741−747. (22) Alam, S. I.; Kumar, B.; Kamboj, D. V. Anal. Chem. 2012, 84, 10500−10507. (23) Conrady, D. G.; Flagler, M. J.; Friedmann, D. R.; Vander Wielen, B. D.; Kovall, R. A.; Weiss, A. A.; Herr, A. B. PLoS One 2010, 5, e15153. (24) Fagerquist, C. K.; Sultan, O. J. Biomed. Biotechnol. 2010, 2010, 123460. (25) Fagerquist, C. K.; Sultan, O. Analyst 2011, 136, 1739−1746. (26) Kitova, E. N.; Daneshfar, R.; Marcato, P.; Mulvey, G. L.; Armstrong, G.; Klassen, J. S. J. Am. Soc. Mass Spectrom. 2005, 16, 1957−1968. (27) Kitova, E. N.; Kitov, P. I.; Bundle, D. R.; Klassen, J. S. Glycobiology 2001, 11, 605−611. (28) Kitova, E. N.; Kitov, P. I.; Paszkiewicz, E.; Kim, J.; Mulvey, G. L.; Armstrong, G. D.; Bundle, D. R.; Klassen, J. S. Glycobiology 2007, 17, 1127−1137. (29) Kitova, E. N.; Mulvey, G. L.; Dingle, T.; Sinelnikov, I.; Wee, S.; Griener, T. P.; Armstrong, G. D.; Klassen, J. S. Biochemistry 2009, 48, 5365−5374. (30) Kondo, F.; Kobayashi, S.; Matsumoto, M.; Yamada, S.; Saito, M.; Suzuki, Y.; Ishikawa, N.; Nakanishi, T.; Shimizu, A. J. Mass Spectrom. 1997, 32, 1140−1142. (31) Kondo, F.; Saito, H.; Hayashi, R.; Onda, H.; Kobayashi, S.; Matsumoto, M.; Suzuki, M.; Ito, Y.; Oka, H.; Nakanishi, T.; Shimizu, A. Analyst 2003, 128, 1360−1364. (32) Meisen, I.; Friedrich, A. W.; Karch, H.; aWitting, U.; PeterKatalinic, J.; Muthing, J. Rapid Commun. Mass Spectrom. 2005, 19, 3659−3665. (33) Domon, B.; Aebersold, R. Science 2006, 312, 212−217. (34) Pan, S.; Aebersold, R.; Chen, R.; Rush, J.; Goodlett, D. R.; McIntosh, M. W.; Zhang, J.; Brentnall, T. A. J. Proteome Res. 2009, 8, 787−797. (35) Picotti, P.; Aebersold, R. Nat. Methods 2012, 9, 555−566.

detect Stx1 at similarly low levels (Figure 3). Lastly we were able to detect and quantify the amount of Stx1a and Stx2a produced by an STEC strain that produced both toxins (Figure 4). In all cases, we were able to detect and quantify toxins in the attomole range.

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CONCLUSIONS We have developed a method of detecting the known Stx2 toxins. In addition, we have demonstrated that we can detect a Stx1 toxin and by analogy detect other variants of Stx1 toxins. Furthermore, we can discriminate between Stx1 and Stx2 toxins as well as discriminate among most of the Stx2 subtypes. Our limit of detection is in the low attomole range, which corresponds to a LOD of approximately 5 fmol/mL in LB. This value is comparable to mouse bioassay, considering that the LD50 for these toxins is 50 ng/kg (∼700 fmol/kg; ∼ 14 fmol/mouse). We have shown that some complex matrices, such as plasma and LB medium, do not contain molecules whose mass spectrometry derived signals would interfere with this approach. Mouse plasma and LB show a matrix effect when low amounts (5 fmol) of toxin are digested in that matrix. The analyte peptides are located in a conserved region of the B subunit, so it is likely that as new Shiga-like toxins are discovered, they will be detectable either directly by this method or by small modifications to it. Lastly, this method of detection does not use intact toxins, so there is no risk of toxin contamination of instruments or toxin exposure to instrument operators. In conclusion, our approach is a safe and effective means of detecting, quantitating, and differentiation among (most of) the known Shiga and Shiga-like toxins from bacterial supernatants in attomole amounts. Further refinement of this method, such as affinity concentration, may extend its utility to clinical samples or other applications.

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ASSOCIATED CONTENT

* Supporting Information S

Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Address: USDA, ARS, 800 Buchanan Street, Albany, CA 94706, United States. E-mail: [email protected]. Tel: (510) 559-6135. Fax: (510) 559-5758. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We wish to acknowledge Dr. Robert Mandrell, Ms. Anna Bates, and Dr. Michael Cooley for kindly providing samples. We wish to acknowledge Mr. Dae Cho for many helpful discussions on instrument optimization.

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REFERENCES

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