Forensic analysis of soman exposure using characteristic fragment ions from protein adducts

Abstract

The verification of exposure to nerve agents is a serious challenge, especially in cases of soman (GD) poisoning. Protein adducts are reliable biomarkers, that provide forensic information and evidence during incidents of terrorism or sporadic poisoning. Mass spectrometry, coupled with a proteomics approach, was established for the forensic analysis of GD-based protein adducts. The fragmentation pathways of GD-based protein adducts were investigated for the first time using electrospray ionization tandem mass spectrometry. Three abundant natural loss product ions, [M+2H-54]²⁺ (loss of two carbon cations), [M+2H-72]²⁺ (loss of tert-butyl and methyl moieties), and [M+2H-84]²⁺ (loss of the pinacolyl moieties), were observed in each of the GD-labeled adducts, and the product ions were independent of protein structure and exposure route. A unique mechanism for the formation of product ions involving GD-protein adducts is proposed here. These findings support the development of a simple and precise forensic analysis technique to rapidly verify GD poisoning using these three GD-related product ions.

Keywords

Adduct metabolites mass spectrometry proteomics soman forensic analysis

Introduction

Soman (Figure 1), O-pinacolyl methylphosphonofluoridate (GD), is a highly toxic organophosphorus chemical warfare agent, classified as a the G-type nerve agent.^1,2 GD is extremely dangerous because it can rapidly and irreversibly inhibit butyryl- and acetyl-cholinesterase after entering the body through respiration or skin penetration.^3,4 Due to its high toxicity and ease of use during terrorist attacks, the development of accurate and specific forensic analysis for GD exposure is essential. Verification techniques are also critical of guiding diagnosis and treatment of GD poisoning in the laboratory, in the environment, and even on the battlefield.^5,6

Figure 1.

The optimized geometry of soman (CAS Reg. No. 96-64-0).

Various methods have been used to detect GD, such as cholinesterase activity,^7–11 dialkyl phosphate metabolites (DAPs),^12,13 and cholinesterase adducts.^10,14–16 Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF MS) has been reported for the detection of adducts from incubates of organophosphorus nerve agent.^17–19 Traditionally, the standard approach used for detecting nerve agent exposure is based on the peptide FGES₁₉₈AGAAS on blood butyrylcholinesterase (BChE); however the brief window for recognition restricts the application of these classic analytical techniques. For this reason, phosphonylated peptides from other proteins with relatively longer half-life, such as albumin,^20–24 are being used as biomarkers to study the effects of GD.^22,25 A considerable body of research has been conducted on the identification of GD-related protein adducts that are based on mass spectrometry,^26–29 but only a few reports have been performed on the cleavage mechanism of GD-based adducts,^30,31 especially information regarding the fragmentation pathways of the electrospray ionization mass spectrum of GD-based protein adducts.

The phosphonylated proteins are extracted from the serum blood by two-dimensional polyacrylamide gel electrophoresis. Upon extraction, the proteins from in vitro and in vivo exposure experiments, using human and rabbit models, are digested using trypsin and analyzed by liquid chromatography-mass spectrometry (LC-MS). As a result, three abundant natural loss product ions were identified. Subsequently, the fragmentation pathways of GD-based protein adducts were investigated. The result of these efforts is a highly accurata and rugger method for detection of GD exposure.

Methods and materials

Chemicals and reagents

GD (>97.0%) was purchased from the Laboratory of Analytical Chemistry, Research Institute of Chemical Defense (Beijing, China). Human albumin (Lot: A1653) and a dye-based blue albumin and immunoglobulin G (IgG) depletion kit were purchased from Sigma-Aldrich. Rabbit albumin (Lot: 170725-0262) was obtained from Equitech-Bio Inc (Kerrville, TX). Wuhan Boster Biological Technology Co., Ltd. provided rabbit IgG (Lot: BST13D01A45). Dithiothreitol (DTT), iodoacetamide (IAM), and 10 kDa MWCO dialysis tubing were obtained from the Boster Company (Wuhan, China). Trypsin (modified, sequencing grade) was purchased from Roche Applied Science (Indianapolis, IN). All other chemicals were purchased from Hanlonda Technology Development Co., Ltd (Beijing, China).

Exposure assays in vitro

Albumin and IgG (100 μL, 1 µg μL⁻¹) in phosphate buffer (pH = 7.4) were exposed to a 100-fold molar excess of GD solution (5 µg μL⁻¹ in isopropanol) for 16 h at 37°C, with shaking. The protein was then denatured by treatment with 8 M urea for 50 min. The denatured protein was reduced by the addition of 50 µL DTT solution (50 mM DTT in 50 mM ammonium bicarbonate (NH₄HCO₃) at room temperature for 1 h and sequentially alkylated using IAM (0.5 M, pH = 8.2 in 25 mM NH₄HCO₃) for 1 h at 25°C in the dark. To remove excess molecules, the samples were placed in 10 kDa MWCO dialysis tubing and dialyzed against 100-fold volume of 10 mM NH₄HCO₃ at 25°C. The buffer was changed every 12 h for 36 h. Proteolysis was further performed in a 25 mM NH₄HCO₃ buffer, with a protein to trypsin ratio of 50: 1, at 37°C for 12 h. Unproteolyzed protein was removed by ultrafiltration at 10,000g for 10 min using an Amicon Ultra 0.5 mL centrifugal filtering device with a molecular weight cut off of 10 kDa (Merck Millipore, Germany). After one washing with 10 mM NH₄HCO₃ buffer, the peptide fraction was collected. The tryptic peptides obtained were lyophilized and stored at −80°C for further mass spectrometric analysis.

Rabbit exposure in vivo

All research using animals (rabbits) was conducted in accordance with the ethics committee of Research Institute of Chemical Defense (Ethical approval number SKLNBC201916).

Prior to each experiment, the rabbits were maintained at an average weight of 2.1 kg and housed in individual cages. Three healthy adult rabbits were injected with a dose of 0.5 times median lethal dose (LD₅₀) of GD via an ear vein (DOTA: LD₅₀ = 13.1 µg kg⁻¹ i.v. in rabbit). Blood samples of 1 mL were collected at 6 h to 14 days after injection. The blood was centrifuged at 1,000g for 5 min at 4°C. The upper layer of serum was collected in clean tubes. Rabbit albumin was extracted from 40 µL of rabbit plasma using a Spin Albumin and IgG Erasin Kit. The immunoglobulin was then separated from the albumin by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The bands corresponding to 50 kDa (IgG heavy chains) and 25 kDa (IgG light chains), identified by Coomassie Blue staining, were excised and subjected to in-gel proteolysis of trypsin.^32,33 The obtained peptide mixtures were identified using mass spectrometry.

MS analysis and proteomics

The peptides produced by proteolysis were analyzed by nano-scale liquid chromatography (LC), coupled with hybrid quadrupole Orbitrap mass spectrometry (Orbitrap Fusion Lumos, Q-Orbitrap-MS). Tryptic peptides (fresh) were dissolved in 10 µL water with 0.1% formic acid (FA). Separation of samples was performed on an UltiMate 3000 nano system (Thermo Scientific, USA). An Agilent analytical column, packed with Zorbax 300SB-C18 (200 mm × 100 µm, 3.0 µm), was employed to perform the separation. The column was maintained at equilibrium for at least 30 min by pumping the starting mobile phase composition prior to injection of phosphonylated peptides. The volume ratios of water/FA/acetonitrile (H₂O/FA/ACN) in mobile phases A and B were 100/0.1/0 and 20/0.08/80, respectively. A linear gradient elution program was applied for separation in 0–120 min with 4–95% mobile phase B. The ion spray voltage was set at 2.5 kV, with a mobile phase flow rate of 500 nL min⁻¹. Xcalibur 2.2 software (Thermo, USA) controlled the mass spectrometer in the data-dependent acquisition manner (DAA). A single full scan (m/z 300–1500) in the Orbitrap MS at a resolution of 70,000 was followed by 10 data-dependent MS/MS scans at normalized collision energy of 27%.

Database search

The labeled sites were searched using the Sequest built-in Proteome Discoverer 2.2 (Thermo, USA) and compared against protein for accurate identification of peptides. Searches were conducted automatically under the following parameters: trypsin was used as the enzyme for protein cleavage, with an allowance of one missed cleavage; carbamidomethylation of cysteine and oxidation of methionine were set as fixed and variable modifications, respectively. Finally, to obtain the covalent modification sites, mass tolerances of precursor and product ions were set at 20 ppm and 0.05 Da, respectively, for all MS/MS spectra. Site probability was calculated for the modified sites to guarantee reliable location information.

Semiquantitative analysis of modified peptides

Approximate quantification of modified peptides at each site was estimated by peak integration, using Xcalibur software. Using the normalization method, the quantitative value of modified peptide was equal to its area under the curve of the extracted ion trace, divided by the total area of all peptides in the sample. Afterward, the values were multiplied by 100,000.

$Peak area intensity = \frac{Area of modified peptide}{Area of all peptides} \times 100, 000$

Results and discussion

MS/MS analysis of the GD-protein adducts

Protein adducts can be detected from the peptide sequences acquired from LC-MS. Peptide sequence and labeled sites were inferred from the parent ions, b and y, as well as the characteristic product ions, which were contained in the secondary spectrum. As shown in Figure 2, the mass of the amino acid residues increased by 162.0810 u (O-pinacolyl methylphosphonate) after phosphonylation by GD. Thus, the GD-labeled sites were inferred by the mass gap. Thirty-three peptides in vitro and five peptides in vivo modified by GD were identified using this method on albumin and IgG in rabbit and human samples, as summarized in Tables S1–S2 in the supplemental data. These active sites involved tyrosine, serine, and lysine. Therefore, these modified peptides constitute a potential family for screening biomarkers after exposure to GD.

Figure 2.

Mass changes in the process of phosphonylation.

Diagnostic ions of GD-IgG adducts

IgG was selected as the target protein to study the GD-protein adducts, which were evaluated in both in vitro and in vivo models using rabbit ear vein injection. The amino acid sequence of the constant region from IgG is relatively stable, making it suitable for retrospective analysis of GD exposure. Considering the constant region of IgG is mainly concentrated on its heavy chain, the phosphonylated peptides on the heavy chain were determined and compared. A total of eight sites on human IgG in vitro and rabbit IgG in vivo were phosphonylated, as shown in Table 1. The peptide VSNK*ALPAPIEK, on human IgG, displayed the strongest signal intensity among the modified sites. Notably, the homologous peptide VHNK*ALPAPIEK also displayed good signal intensity under GD exposure in rabbits. These data indicated that the peptide has the potential to become a biomarker for traceability research of GD-based exposure during a terrorist event.

Table 1.

GD-modified peptides on IgG in vitro and in vivo.

NO.	IgG species^a	Sites^c	Labeled peptides	Peak area intensity
1	Human IgG in vitro	Y298	NWY*VDGVEVHNAK	23.76
2		S259	LGGPS*VFLFPPKPK	0.63
3		S154	FPLAPSSKS*T	2.51
4		S344	VSNK*ALPAPIEK	33.23
5	Rabbit IgG in vivo^b	Y304	VY*TMGPPR	3.75
6		Y278	FTWY*INNEQVR	2.31
7		S370	LSVPTS*EWQR	24.58
8		K281	VHNK*ALPAPIEK	44.76

^a The NCBI accession numbers of human IgG and rabbit IgG are AAB86467.2 and AAA64252.1, respectively.

^b An injection dose of 0.5 × LD50.

^c The amino acid sequences does not include signal peptides of proteins.

Figure 3 shows the representative MS/MS spectra of the homologous GD-IgG peptides of different species obtained from in vivo and in vitro exposure experiments, respectively. The VSNK*ALPAPIEK peptide from human IgG exposure in vitro (Figure 3a) and the VHNK*ALPAPIEK from rabbit exposure in vivo (Figure 3b) differed in the second position from the N terminus. Three key product ions were observed, including precursor ions in the green line, y ions in the blue line, and b ions in the red line. The precursor ions ([M+2H]²⁺-H) confirmed that one GD molecule was added to the peptides. The y₁–y₈ and b₂–b₃ ions demonstrated that the amino acid sequences of VSN/VHN and ALPAPIEK were not phosphonylated, inferring that the fourth lysine from the C terminus was labeled on these two peptides.

Figure 3.

MS/MS spectra and magnified image spectra of GD-labeled adduct on IgG. (a) Adduct in VSNK*ALPAPIEK peptide in vitro exposure. (b) Adduct in VHNK*ALPAPIEK peptide in vivo exposure. (c) Magnified view of neutral loss product ions from precursor ions in VSNK*ALPAPIEK peptide. (d) Magnified view of neutral loss product ions from precursor ions in VHNK*ALPAPIEK peptide.

Interesting fragmentation peaks were obtained, involving the appearance of three neutral loss product ions from precursor ions, [M+2H-54]²⁺, [M+2H-72]²⁺, and [M+2H-84]²⁺, marked in the green lines in Figure 3. These product ions were not found in other G-type protein adducts, such as sarin, cyclosarin, tabun, and VX. Most importantly, the product ions observed all had high intensity. To illustrate the three neutral loss product ions, the portions marked by the dashed rectangle in Figures 3a and 3b were magnified and shown in Figures 3c and 3d, respectively. The three neutral loss product ions, [M+2H-54]²⁺, [M+2H-72]²⁺, and [M+2H-84]²⁺, were clearly visible, and it was evident that each neutral loss peak had three to four isotope peaks, which were separated by m/z 0.5. Therefore, this group of characteristic ions can be considered indicators of GD exposure to IgG in vitro and in vivo.

Diagnostic ions of GD-albumin adducts

To investigate whether the previously discovered GD-based diagnostic ions were involved in the types of proteins, research was conducted on the GD-albumin adducts. Similar findings were observed in the MS/MS spectra of GD-labeled peptides on rabbit albumin (UniProt accession number: G1U9S2), as shown in Figure 4. The spectra contained GD-tyrosine and GD-lysine adducts. In Figure 4a, key ion product ions b₂ and y₁–y₁₀ were observed, demonstrating the presence of characteristic ions of GD in the K*QTALVELLK peptide. The difference between the mass at y₁₀ (1014.6278 u) and the mass at y₉ (888.5668 u) equaled the mass of the lysine residue (128.0914 u) plus the O-pinacolyl methylphosphonate from GD (162.0810 u), inferring that K525 was phosphonylated at position eight from the C terminus. Like the rule of the GD-IgG adduct, a set of product ion peaks (the loss of 84.0432 u, 72.0448 u, and 54.0314 u) with strong m/z signals and fixed intervals are shown in Figure 4b. The gap between y₆ and y₅ proved that the tyrosine of the peptide AY*EATLK was modified by GD in Figure 4c. The three high-abundance product ions (the loss of 84.0946 u, 72.1028 u, and 54.0874 u) are shown in Figure 4. The presence of these product ions suggests that the diagnostic ions were formed from the GD-tyrosine adduct. These data indicate that the diagnostic ions, [M+2H-84]²⁺, [M+2H-72]²⁺, and [M+2H-54]²⁺, are related to GD exposure, independent of the nature of the proteins.

Figure 4.

MS/MS spectra of GD-labeled rabbit albumin-adducts. (a) Adduct in K*QTALVELLK peptide in vivo exposure. (b) Neutral loss product ions from precursor ions in K*QTALVELLK peptide. (c) Adduct in AY*EATLK peptide in vivo exposure. (d) Neutral loss product ions from precursor ions in AY*EATLK peptide.

Fragmentation pattern of diagnostic ions of GD-based adducts

A fragmentation pathway for GD-protein adducts was proposed to explain the observed characteristic peaks. As shown in Figure 5a, the precursor ions were fragmented into the neutral loss peaks of [M+2H-84]²⁺ via expulsion of the pinacolyl group in the MS/MS spectra. A mechanism of the McLafferty rearrangement was proposed, which involves hydrogen migration (1,5 C─O H shift) to the oxygen. This conclusion was based upon the hydrolysis concept of other G-type nerve agents, such as sarin.³⁴ Product ions of [M+2H-72]²⁺ were generated due to the cleavage of tertiary carbon from the precursor ions, undergoing two steps in the loss of the carbon cation, as shown in Figure 5b. If, after loss of a carbon cation, the radical cation can perform a random rearrangement of hydrocarbons³⁵ and rearrangement of two hydrogen atoms of phosphate esters,³⁶ then product ion [M+2H-54]²⁺ will form, as shown in Figure 5c. Obviously, formation of these three product ions is related to the pinacolyl group, yielding to the prominent characteristic ions of GD exposure. Thus, the proposed pathway of the GD-based adducts could be useful for rapid identification and analysis of GD-related nerve agents.

Figure 5.

Fragmentation pathway of GD-peptide adducts. (a) Production process of [M+2H-84]2+ ions. (b) Production process of [M+2H-72]2+ ions. (c) Production process of [M+2H-54]2+ ions.

Conclusion

This paper describes the use of a Q-Orbitrap-MS with standard proteomics methodologies to determine GD-labeled adducts following GD exposure in vitro and in vivo. Data showed that several binding sites in albumin and IgG could be used for retrospective detection of GD poisoning. For the first time, we propose a pathway for the fragmentation of GD-protein adducts. Three GD-related characteristic product ions with high intensity, [M+2H-54] ²⁺, [M+2H-72]²⁺, and [M+2H-84]²⁺, were observed. Based on these product ions, a reliable, precise, simple, and rapid forensic analysis technique can be developed for GD poisoning.

Supplemental material

Supplemental Material, sj-pdf-1-het-10.1177_09603271211001111 - Forensic analysis of soman exposure using characteristic fragment ions from protein adducts

Supplemental Material, sj-pdf-1-het-10.1177_09603271211001111 for Forensic analysis of soman exposure using characteristic fragment ions from protein adducts by F Fu, Y Guo, X Lu, P Zhao, S Zou, H Wang, R Gao and C Pei in Human & Experimental Toxicology

Footnotes

Authors’ note

In this article, the binding sites of GD on albumin and IgG in rabbit and human samples, as well as the MS/MS spectra of the other GD-labeled peptides, appear in the Supplementary Data, which can be found in the online version.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was performed with the financial support of the State Key Laboratory of NBC Protection for Civilian (No. SKLNBC2019-10).

ORCID iD

R Gao

Supplemental material

Supplemental material for this article is available online.

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