Proteinaceous toxins are harmful proteins derived from plants, bacteria, and other natural sources. They pose a risk to human health due to infection and also as possible biological warfare agents. Paper spray mass spectrometry (PS-MS) with wipe sampling was used to detect proteins from surfaces as a potential tool for identifying the presence of these toxins. Proteins ranging in mass between 12.4 and 66.5 kDa were tested, including a biological toxin simulant/vaccine for Staphylococcal enterotoxin B (SEBv). Various substrates were tested for these representative proteins, including a laboratory bench, a notebook cover, steel, glass, plant leaf and vinyl flooring. Carbon sputtered porous polyethylene (CSPP) was found to outperform typical chromatography paper used for paper spray, as well as carbon nanotube (CNT)-coated paper and polyethylene (PE), which have been previously shown to be well-suited for protein analysis. Low microgram quantities of the protein toxin simulant and other test proteins were successfully detected with good signal-to-noise from surfaces using a porous wipe. These applications demonstrate that PS-MS can potentially be used for rapid, sample preparation-free detection of proteins and biological warfare agents, which would be beneficial to first responders and warfighters.
The first modern documented use of biological warfare agents (BWAs) dates back to at least the mid-eighteenth century when British troops distributed smallpox-contaminated blankets to Native Americans during the French and Indian War . During the early twentieth century, many nations had active biological weapons programs including the USA (ending in 1972) and the Soviet Union (ending in 1992) . Programs from both nations successfully weaponized multiple agents, including toxins. Since 1972 , 170 nations have signed the Biological and Toxin Weapon Convention prohibiting the research and development, production, and stockpiling of BWAs. Despite these efforts, the convention has no oversight or enforcement mechanism, making it difficult to police participating nations. Perhaps more concerning is the continued development of this weapon class by rogue nations and extremist groups. In fact, between 1990 and 2011, there were 227 known incidences involving a bio-agent attack . The impact and difficulty in stopping these attacks was never more evident than the 1984 attack in The Dalles, Oregon, perpetrated by the Bhagwan Shree Rajneesh cult. From this attack, 751 people were poisoned by consuming food from salad bars at 10 different restaurants, which were intentionally contaminated with Salmonella cultures. It took nearly two years for authorities to determine that this event was an incident of bioterrorism [4, 5]. More recently, in 2001, letters containing powdered anthrax spores were mailed to numerous locations including U.S. Senate offices and news outlets [6, 7]. In modern times, a large percentage of bio-agent attacks have involved toxins, in particular, ricin . It is widely believed that its popularity is largely due to the ease by which beans from the castor oil plant, Ricinus communis, can be legally obtained .
Since the anthrax letter attacks, the ability to detect biological threats has advanced significantly [9,10,11]. Lateral flow immunoassays are commonly used for rapid presumptive detection of biological threats . However, these antibody-based assays have several drawbacks that include limited shelf life, temperature sensitivity, and high rates of false positives and negatives. For confirmatory analyses, a variety of technologies based on polymerase chain reaction [13, 14] and isothermal amplification  are available, some of which are field-deployable for first responders and warfighters. A variety of next-generation sequencing technologies can be used to rapidly sequence genomes and identify biothreats, including unknowns and genetically modified organisms . Lab-based sequencing instruments from Illumina, Pacific Biosciences, and others have become well-established. Recently, a portable genomic sequencer called the MinION was introduced by Oxford Nanopore Technologies [17, 18]. The size, weight, and power requirements were greatly reduced by the utilization of a new technology called nanopore sequencing [19, 20].
Even though toxins are protein-based and do not contain DNA, they can still be identified by DNA amplification and genomic sequencing technologies for samples containing co-purified DNA from the organism that produced the toxin. For instance, ricin can be presumptively identified if a suspicious sample contains DNA from Ricinus communis [21, 22]. Lateral flow immunoassays can also be used to identify biological toxins, but along with DNA amplification technologies, these assays are not threat agnostic.
The analysis of biowarfare agents by mass spectrometry (MS) has grown substantially in recent years, but to date is still largely limited to brick and mortar laboratories equipped with analytical grade instrumentation and supporting equipment . Nearly all of the BWA methods require extensive sample preparation and lengthy liquid chromatography (LC) methods . Alternative near prepless ambient ionization techniques such as direct analysis in real time (DART) MS, proton transfer reaction (PTR) MS, and desorption electrospray ionization (DESI) MS have all been proposed to overcome the limitations of traditional LC-MS/MS identification pipelines. Of these approaches, DESI has shown the most potential as it is able to ionize macromolecules such as proteins [25, 26]. However, the DESI process still requires the use of analytical pumps and compressed gas. There are also size and shape constraints on the sample due to the need for the substrate to be close to the mass spectrometer and the DESI source. BWAs have also been analyzed by biomolecular interaction analysis (BIA) MS  and matrix-assisted laser desorption/ionization (MALDI) MS . However, these methods, like the LC-based methods, require extensive sample preparation and are not well-suited for field use.
Like DESI, paper spray ionization (PS) MS is also amendable to the analysis of small molecules and macromolecules alike. This technique has been extensively demonstrated for a wide array of applications, especially for small molecules such as pharmaceutical, illicit drugs, and chemical warfare agent surrogates [29,30,31]. In most cases, the PS-MS technique requires minimal sample preparation  and can be performed in less than a minute from complex biological and environmental samples [29, 30, 33,34,35,36]. Several recent studies have demonstrated improved detection of intact proteins by PS-MS through a number of creative paper substrate modifications including the addition of carbon nanotubes (CNT) [37, 38] and silica particles . The use of alternative materials for protein analysis, such as size exclusion membranes , porous polymer wicks , and CNT-coated polyethylene , has also been reported.
In this study, proteins were analyzed from a variety of surfaces by combining paper spray mass spectrometry with wipe collection. Several proteins ranging in size from 12.4 to 66.5 kDa were tested. Analysis of a protein toxin simulant, Staphylococcal enterotoxin B vaccine (SEBv) , is also demonstrated. The data is intended to demonstrate proof of concept for the detection of biological warfare agent simulants by paper spray mass spectrometry from wiping of contaminated surfaces.
Chemicals and Materials
High-performance liquid chromatography (HPLC) grade solutions of methanol, acetonitrile, formic acid, and water were purchased from Sigma-Aldrich (St. Louis, MO, USA). HPLC grade acetic acid was purchased from Fisher Scientific (Hampton, NH, USA). SEBv was made in-house at ECBC, as previously described , and stored in 1X PBS. Lysozyme and cytochrome c were purchased from Sigma-Aldrich. Velox sample cartridges were purchased from Prosolia (Indianapolis, IN, USA). The porous polyethylene (PE) substrate (7–12 μm pore size, 0.6 mm thick) was purchased from Porex (#XS-POR-7744, Fairburn, GA, USA), and polycarbonate track-etched membranes were purchased from Whatman, Inc. (Florham Park, NJ, USA). Single-walled CNTs were purchased from Sigma-Aldrich (704113-250MG). The porous nylon membrane with 5 μm diameter pores used for wiping was purchased from Sterlitech (NY5025100, Kent, WA, USA). Cyclopore track-etched membranes (5 μm pore size, 25 mm thick) were purchased from Whatman, Inc. Carbon rods for sputter coating were purchased from SPI Supplies (West Chester, PA, USA).
CNT-Coated PE Preparation
The CNT-coated spray substrates were prepared as previously described [37, 42]. Briefly, the PE was cut into rectangles (2.0 cm × 0.5 cm), washed by immersion in pure methanol, vortexed for 1 min (3X), washed by immersion in 1:1 methanol:water, and vortexed for 1 min (3X). The thin piece of PE was placed on an absorbent pad, and the CNT slurry (10 mg/mL in methanol) was added onto the PE. The CNT slurry was allowed to dry and then washed again, as described above. The CNT-coated PE was allowed to dry then cut into spray tips. To form the spray tip, one side of the rectangle (short side) was cut evenly to a fine triangular point. Spray tips were examined under a microscope and any exhibiting fraying or other signs of damage were omitted.
Carbon Sputtered Porous PE Preparation
A 3″ × 3″ sheet of porous PE was coated with carbon to a thickness in excess of 100 nm using a Desk V sputtering system (Denton Vacuum, Moorestown, NJ, USA). With this system, two sharpened carbon rods are held in contact via tension applied by a metal spring, and the system is pumped down to at least 10−4 torr prior to deposition to allow for a uniform coating on the PE substrate, due to decreased path length. Once the appropriate pressure is reached, a current of 15–30 A is applied to the carbon rods to achieve deposition. The current is controlled manually and must be monitored to obtain reproducible deposition rates, especially without the aid of a deposition rate monitor. Since the carbon rods only provide approximately 25–30 nm thick layers, the deposition is repeated until a thickness of at least 100 nm is reached, which provides a uniform coating that remains wettable by methanol:water spray solvents. Thickness of carbon layers is determined by depositing carbon on a thin layer of gold coated on a glass substrate and observing the color change. A color shift from gold to purple is observed for carbon thicknesses ~ 200–250 Å . The PE sheet was cut into 0.5 cm wide strips and sonicated in methanol for 10 min to remove any loose carbon particles then allowed to air dry. Once dried, the strips were cut to 1.5 × 0.5 cm rectangles and a triangular tip (~ 33°) was cut from the 0.5 cm side, resulting in a pentagonal-shaped spray substrate. Since the spray substrates are reusable, the tips were rinsed with methanol and 5% formic acid in water then dried prior to the subsequent experiment. When not in use, the tips can be stored in 5% formic acid in water until needed.
Sample Deposition and Wiping
Stock protein solutions were diluted in water, and 10 μL was spotted onto the substrate and allowed to dry for 1 h. This was sufficient time for all water to evaporate, leaving a dried protein spot. To effectively wipe the dried protein spots off the surface, 5 μL of 5% formic acid in water was added to the dried spots and wiped with a 5 mm diameter porous membrane disk (5.0 μm pore size) for 30 s, and the membrane was allowed to dry on the surface. Spotting solvent on the dried protein spot was chosen to avoid dilution and spreading of the protein as well as to better control the amount of wipe solvent present. This was found to improve collection efficiency of the protein off the surface. Once completely dry (~ 15 min), the membrane was ready to be analyzed by paper spray mass spectrometry.
The membrane, containing dried protein wiped from a surface, was placed in contact with the carbon sputtered porous polyethylene (CSPP) substrate in a machined Delrin cartridge (Figure 1A, B), designed to hold and provide electrical contact to the spray tip. A total volume of 60 μL spray solvent (3:1 methanol:water, 1% acetic acid) was added dropwise to the spray substrate using a micropipette over a period of about 10 s. A spray voltage between 3.5 and 4 kV was applied for ionization. The spectra were collected and averaged for 1 min.
Mass Spectrometry Data Acquisition and Processing
The protein detection work was performed on a Thermo Q-Exactive Focus mass spectrometer (Thermo Scientific Inc., San Jose, CA, USA). The resolution was set at 17,500 with 10 microscans. The spray voltage was between 3.5 and 4 kV, the S-lens voltage was 50 V, and the in-source collision-induced dissociation (CID) was 22.0, 25.0, 15.0, and 25.0 V for myoglobin, cytochrome c, carbonic anhydrase, and bovine serum albumin (BSA), respectively. Deconvolution of the protein spectra was performed using MagTran V1.03b2 . Calibration curves were obtained from 1/x2 weighted least squares.
Results and Discussion
As shown in Figure 2A, like other studies , this work confirmed that chromatography paper showed poor sensitivity for intact lysozyme (7 nM, dissolved in the spray solvent). Chromatography paper and a porous PE substrate, both coated with CNT's [37, 42], were also investigated as alternative spray substrates in addition to a new spray substrate: CSPP. As shown in Figure 2B and Figure 2C, sensitivity improved significantly for both CNT-coated substrates relative to chromatography paper. The signal intensity obtained during the analysis of the lysozyme solution using the CNT-coated porous PE was approximately 20 times higher than CNT-coated paper and 100 times higher than uncoated chromatography paper. In comparison to the CNT-coated PE, the CSPP substrates exhibited roughly the same signal intensity, but signal-to-noise improved by more than an order of magnitude, as shown in Figure 2D. Additionally, solvent clustering and adduct ions are visibly lower for the CSPP compared to the paper and CNT-coated substrates. We hypothesize that the improved signal intensity of the carbon-coated PE substrates arises from smaller droplet sizes generated during the ionization process owing to the smaller size of the Taylor cone generated from these substrates  and the slower solvent flow rate. The lower baseline, as well as the lower intensity of protein adduct ions, may arise from the improved surface coverage of the carbon sputtered material.
Sputter deposition is a widely employed technique to deposit thin films of pure metals, alloys, and other conductive materials onto a substrate for a variety of uses including electron microscopy sample preparation , micro- to nanoscale electrodes [48, 49], and micro- and nanofabrication . Here, porous PE was coated with carbon using a commercially available carbon rod evaporation accessory designed to work with the sputtering system. SEM images of porous PE coated with CNT or CSPP are shown in Figure 1C, D, respectively. There is a noticeable improvement in the coating uniformity, and the porous nature of the surface is better preserved with the carbon sputtered surface. Unlike the CNT-coated substrates, the CSPP substrate also exhibited no signs of surface charging during SEM. The lack of charging for CSPP suggests more complete coverage of the polymer substrate.
Compared to the more manual method for preparing CNT-coated PE substrates , the carbon sputtered substrates were prepared without manual steps, and the amount prepared in a given batch is limited only by the sputter chamber size. Due to fewer cleaning steps, faster sample preparation, improved signal-to-noise intensity, and more uniform coverage than that of the CNT coating method, the carbon sputtering method was chosen for all protein analyses. Individual CSPP spray tips were reused in excess of 50 times each with no noticeable signal degradation or carry over during this interval.
Demonstration of Wiping Proteins from Surfaces
Cytochrome c, myoglobin, carbonic anhydrase, and BSA were wiped from a glass slide and analyzed using the CSPP substrate. Molecular weights, isoelectric points, and limits of detection (LODs) of these proteins are shown in Table 1. Limits of detection were determined for myoglobin and cytochrome c using linear calibration curves (Figure 3) by multiplying the standard error of the y-intercept by three and dividing by the slope. For carbonic anhydrase and BSA, the LODs were estimated from the highest successfully detected calibrator because there were not enough points to form a reasonable line. Mass spectra obtained for these proteins wiped from glass are shown in Figure 4. As shown in Figure 4C, BSA exhibited a low signal-to-noise compared to the other three proteins. This is likely due to the poor mass spectral response of larger proteins obtained with ESI, which is well-known [51,52,53]. In addition to abundant peaks from the proteins, background peaks from the nylon membrane or other polymers were present. These included m/z 927.66, a cyclic oligomer of polyamide-66 with ionic formula [C48H88N8O8+Na]+ from the nylon membrane. Another abundant background peak was m/z 803.54, likely a sodium-bound dimer of diisooctyl phthalate, a ubiquitous plasticizer .
To further examine protein wiping efficiency, cytochrome c was spotted on various untreated and uncleaned common laboratory surfaces and analyzed, as described above. Here, 10 μL of 1 mg/mL cytochrome c was spotted on six different surfaces: laboratory benchtop, a textured plastic chair seat, leaf from the rubber plant (Ficus elastica), cardboard notebook cover, stainless steel scissors, and a vinyl rubber wall base (Figure 5). Absorbent surfaces were avoided because they are less suitable for wipe sampling. Care must be taken when wiping off rough surfaces, such as the textured plastic chair seat, to avoid tearing. Mass spectral peaks arising from cytochrome c were obtained from each of the surfaces. Some of the surfaces, such as the cardboard notebook cover and wall board, exhibited more cluster peaks around the main molecular ions of the protein, presumably due to higher amounts of adventitious sodium and other cations. Additionally, the spectral intensity varied somewhat among the surfaces, although it is not clear if this is caused by ion suppression or surface effects on protein recovery.
To assess repeatability, five technical replicates of cytochrome c (10 μg) were analyzed off a glass substrate using the described wiping protocol. The relative standard deviation was determined to be 8.2% by calculating the area under the curve for the 1030 m/z peak. The variance will depend on several factors including surface properties, the protein, and wiping technique.
Toxin Simulant Wiping
The coated PE substrate was next used to analyze the toxin simulant SEBv. This protein is structurally homologous to the SEB toxin, and the primary amino acid sequence differs at three amino acids . The recombinant protein used here also contained a polyhistidine-tag at the N terminus. A mass spectrum of SEBv was first obtained from infusion via electrospray ionization (Figure 6A). The deconvoluted mass spectrum indicated a mass for SEBv of 30,257 Da (Figure 6B), which was consistent with the molecular mass of the protein predicted from the expression vector (30.3 kDa) as well as an SDS-PAGE gel on the same isolated protein (data not shown). A DC offset of 28–30 V was applied in the source to aid in protein desolvation and adduct removal; this potential also resulted in some in-source CID of the proteins. An advantage of using this fragmentation is that the selectivity of protein detection is improved without the need for performing tandem mass spectrometry.
Typical exposures of 5–20 μg orally and 20 to 500 μg intravenously have shown to induce emesis in nonhuman primates . In humans, the estimated 50% lethal dose (LD50) was estimated to be 0.02 μg/kg via aerosol exposure, whereas ocular exposure was nonlethal but symptomatic around 50 μg . To investigate the potential for detecting biological toxins from surfaces at the low μg level, 10 μL of a 1 mg/mL solution of SEBv was applied to a laboratory bench (10 μg total protein) and allowed to dry. The dried SEBv spot (Figure 6C) was wiped with a small piece of nylon membrane wetted with a 1:1 methanol:water solution. The nylon membrane was then placed on top of the spray substrate followed by addition of spray solvent and ionization voltage. The charge distribution was different for the wiped SEBv compared to the spectrum obtained from ESI infusion of the protein standard, but the mass was the same between the two experiments. Differences in the charge distribution may be caused in part by the amount of acid present: 1% acetic acid in the spray solvent compared to the 5% formic acid solution used for protein wiping. The spray substrate itself may also affect the charge distribution as suggested by the results in Figure 2. More experiments are required to determine the variables affecting protein charge distribution.
The work detailed in this study was performed with single-use nylon membrane punches; however, actually applying this technique in the field could be challenging and cumbersome. Therefore, we designed a new reusable sampling pen depicted in Figure 7. In this conceptual design, the pen is filled with any tubular porous material that can be used to sample a surface. Once the sample has been acquired, the tip of the material can then be cut off to form a disk which can be placed onto the paper spray cartridge for analysis. Collectively, this system simplifies the sampling process and also provides for a reusable platform for interrogation of multiple suspect surfaces.
This work demonstrates the feasibility of analyzing simulants of biological warfare agents and other proteins from surfaces using paper spray MS coupled to surface wiping. A novel alternative substrate consisting of CSPP demonstrated improved sensitivity compared to traditional chromatography paper or CNT-coated paper and PE. This technique allowed us to detect the biological toxin simulant, SEBv, from a contaminated surface (i.e., laboratory bench). Further experiments need to be conducted to demonstrate PS-MS’s ability to analyze other biological threats (e.g., bacteria, viruses).
The toxin simulant used in this study is intended to demonstrate the potential use of paper spray MS to detect protein toxins. We previously demonstrated the use of paper spray ionization for the analysis of a variety of other threat agents including chemical warfare simulants and hydrolysis products [31, 36]. Taken together, this work demonstrates that a single near prepless ambient ionization technique is capable of detecting both chemical and biological threats. This technique could have immediate utility in field-forward detection facilities with laboratory-scale mass spectrometers. One such example is the US Army’s CARA mobile expeditionary laboratories, which consist of modular, semi-mobile laboratories equipped with liquid chromatography-mass spectrometry systems . Another is the US Army’s JUPITR program, in which orbitrap mass spectrometers (among others) are deployed . While such programs employ laboratory-scale instrumentation, they would nevertheless benefit from faster and simpler methods for MS-based protein detection. Another important step to enable rapid field analysis of toxins is use of portable mass spectrometers, which have been coupled to paper spray by several research groups [57,58,59,60]. Protein detection on portable mass spectrometers has also been demonstrated [61,62,63], although the lower resolution, limited sensitivity, and smaller mass range of portable instruments will naturally constrain the detection of intact proteins.
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This work was supported by a grant (CB10238) from the Joint Science and Technology Office (JSTO) and the CBA Division of the Defense Threat Reduction Agency (DTRA). DTRA is a Combat Support Agency and a Defense Agency with a three-pronged mission: (1) to counter the threats posed by the full spectrum of weapons of mass destruction (WMD), including chemical, biological, radiological, nuclear, and high-yield explosives; (2) counter the threats posed by the growing, evolving categories of improvised threats, including improvised explosive devices, car bombs, and weaponized consumer drones, as well as the tactics, technologies, and networks that put them on the battlefield; (3) ensure the U.S. military maintains a safe, secure, effective, and credible nuclear weapons deterrent.
The authors would like to thank Alena Calm (CCDC Chemical Biological Center) for kindly providing all purified SEBv used in this study. The authors would also like to thank the Integrated Nanosystems Development Institute (INDI) for the use of the sputtering and SEM systems, as well as David Heemstra (NDnano) at the University of Notre Dame for his contribution and valuable discussions regarding thin film deposition. Conclusions and opinions presented here are those of the authors and are not the official policy of the US Army, CCDC Chemical Biological Center, or the US Government. Information in this report is cleared for public release and distribution is unlimited.
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Wichert, W.R.A., Dhummakupt, E.S., Zhang, C. et al. Detection of Protein Toxin Simulants from Contaminated Surfaces by Paper Spray Mass Spectrometry. J. Am. Soc. Mass Spectrom. 30, 1406–1415 (2019). https://doi.org/10.1007/s13361-019-02141-6
- Large molecule
- Ambient ionization
- Direct analysis
- Cartridge prototyping