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Protein adduct binding properties of tabun-subtype nerve agents after exposure in vitro and in vivo
Toxicology Letters 321 (2020) 1–11
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Protein adduct binding properties of tabun-subtype nerve agents after exposure in vitro and in vivo
T
Feiyan Fu, Haibo Liu, Runli Gao, Pengcheng Zhao, Xiaogang Lu, Ruihua Zhang, Liangliang Wang, Hongmei Wang*, Chengxin Pei* State Key Laboratory of NBC Protection for Civilian, Beijing 102205, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Nerve agents Tabun Albumin Q-Orbitrap Lysine adducts
Upon entering the body, nerve agents can bind active amino acid residues to form phosphonylated adducts. Tabun derivatives (O-alkyl-N,N-dialkyl phosphoroamidocyanidates) have strikingly different structural features from other G-series nerve agents, such as sarin and soman. Here, we investigate the binding mechanism for the phosphonylated adducts of nerve agents of tabun derivatives. Binding sites for three tabun derivatives, O-ethylN,N- dimethyl phosphoramidocyanidate (GA), O-ethyl-N,N-ethyl(methyl) phosphoramidocyanidate, and Oethyl-N,N-diethylphosphoramidocyanidate were studied. Quadrupole-orbitrap mass spectrometry (Q-OrbitrapMS) coupled to proteomics was used to screen adducts between tabun derivatives and albumin, immunoglobulin, and hemoglobin. The results reveal that all three tabun derivatives exhibit robust selectivity to lysine residues, rather than other amino acid residue types. A set of 10 lysine residues on human serum albumin are labeled by tabun derivatives in vitro, with K525 (K*QTALVELVK) and K199 (LK*CASLQK) peptides displaying the most reactivity. Tabun derivatives formed stable adducts on K525 and K414 (K*VPQVSTPTLVEVSR) for at least 7 days and on K351 (LAK*TYETTLEK) for at least 5 days in a rabbit model. Three of these peptides—K525, K414, and K351—have the highest homology with human serum albumin of all 5 lysine residues that bound to examined rabbit blood proteins in vivo. Molecular simulation of the tabun-albumin interaction using structural analysis and molecular docking provided theoretical evidence supporting lysine residue reactivity to phosphonylation by tabun derivatives. K525 has the lowest free binding energy and the strongest hydrogen bonding to human albumin. In summary, these findings identify unique binding properties for tabun derivatives to blood proteins.
1. Introduction Nerve agents (NAs) (Bajgar, 2004a) have been used since the World War II and are toxic and lethal chemical weapons that produce harmful effects on human health. After World War II, many hundred thousand tons of chemical warfare agents (CWA) were destroyed at sea. Among them, the coastal areas of Europe, Russia, Japan, and the United States have been affected by nerve agents, sulfur mustard, and Louise appearing to be the most common CWA disposed at sea. Since then, the international community has made it a priority to address the related environmental risks. Also, even if the use and acquisition of NAs is prohibited under the Chemical Weapon Convention (CWC) of 1993(1997)Chemical Weapon Convention (CWC) of, 2019Chemical Weapon Convention (CWC) of 1993(1997), NAs could be used by terrorists today that would pose a threat to human health and peace
(Bhaganagar and Bhimireddy, 2017; Nakagawa and Tu, 2018; Nepovimova and Kuca, 2018). Therefore, the search for better ways to quickly detect and monitor NAs has major importance for human safety and security, particularly for verification and traceable analysis in human poisoning events. The most commonly utilized method is the analysis of dialkyl phosphate metabolites (DAPs) and other metabolites in urine (Black et al., 1994; Shafik and Enos, 1969). However, these metabolites have half-lives ranging from 24 to 48 h, rendering this method futile for longterm detection of NAs in human poisoning events (Pardasani et al., 2007). Therefore, NA-protein adducts are potentially beneficial for tracing NA exposure since they have longer half-lives than DAPs and other metabolites related NA exposure (Black et al., 1999; Lee et al., 2018; Lockridge and Schopfer, 2010; Pardasani et al., 2007; Read et al., 2010; Schmidt et al., 2014; Schopfer and Lockridge, 2012).
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Corresponding authors at: State Key Laboratory of NBC Protection for Civilian, Beijing, 102205, China. E-mail addresses: [email protected] (F. Fu), [email protected] (H. Liu), [email protected] (R. Gao), [email protected] (P. Zhao), [email protected] (X. Lu), [email protected] (R. Zhang), [email protected] (L. Wang), [email protected] (H. Wang), [email protected] (C. Pei). https://doi.org/10.1016/j.toxlet.2019.12.014 Received 2 August 2019; Received in revised form 9 December 2019; Accepted 13 December 2019 Available online 14 December 2019 0378-4274/ © 2019 Published by Elsevier B.V.
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kg−1, 400 μg kg -1, and 800 μg kg -1, respectively, i.v. in female mice). Personal protection is required against poisoning during all experiments.
Accordingly, a few proteins in blood plasma, such as cholinesterase, albumin, and hemoglobin, have become useful protein biomarkers for nerve agent exposure (Chen et al., 2013; Gupta et al., 2005; John et al., 2010; Peeples et al., 2005). The standard method used for biomarkers of nerve agent exposure is currently based on nine peptides on blood butyrylcholinesterase (BChE); however, the purification of NAs-ChE adducts is problematic. Albumin possesses advantages as a biomarker of exposure compared with the serine active on BChE. Tyrosine adducts are more stable and do not age like serine active sites on BChE. On the other hand, albumin present in much larger quantities compared with BChE. Further, albumin is present in much larger quantities compared with BChE. Thus, high abundant blood proteins may be more useful biomarkers compared with those from BchE. The G-series—which includes sarin (GB), soman (GD), and tabun (GA)—is one of the most important classes of nerve agents. O-ethylN,N- dimethyl phosphoramidocyanidate (GA) (Gupta et al., 1987; Heilbronn and Tolagen, 1965) belongs to the G-series nerve agents and produces acute toxicity on the nervous system through acetylcholinesterase (AChE) inhibition, quickly resulting in paralysis, seizure, and death (Bajgar, 2004b; Carletti et al., 2010; Mercey et al., 2012; Pardio et al., 2001; Schecter, 2004). In the 1980s, more than 400 people were poisoned in a chemical attack in the Iran-Iraq war, which has been investigated for the GA use (Haines and Fox, 2014). As a primary nerve agent, the structure of GA is more specific than the other NAs in the G-series family. The PeCN and PNe bonds in GA, which are different from the PeF and PCe bonds in sarin and soman (see Fig. 1), make the phosphonylation reactivity of GA with proteins distinctive. Covalent adducts are formed between nerve agents and proteins via phosphonylation—a nucleophilic substitution reaction. The existence of a lone electron pair located on the ortho-functional group (N,N-dialkyl) makes the nucleophilic attack of GA different from sarin and soman. On the other hand, since the leaving ability of CN- in GA is slightly weaker than the F- in sarin and soman, the nucleophilic reaction process for adduct formation is affected. Here, we examine the selectivity and phosphonylation of active sites on proteins by GA. We used three nerve agents (Fig. 2), GA, O-ethyl-N,N-ethyl(methyl) phosphoramidocyanidate (MEGA), and O-ethyl-N,N-diethylphosphoramidocyanidate (EEGA), which contain the same chemical structural framework, to represent GA derivatives (GAs). Three blood proteins—albumin, hemoglobin, and immunoglobulin (IgG) —were used to reveal the phosphonylation mechanism of GAs. To evaluate the binding selectivity of GAs to proteins, we performed exposure assays of GAs with proteins in vitro, plasma in vitro, and rabbit in vivo. The adducts bound by GAs were identified using quadrupole-Orbitrap mass spectrometry (Q-Orbitrap) coupled with nanoscale liquid chromatography method (nLC). Furthermore, molecular simulation studies examined the interaction between GA and albumin lysine residues. The results show that GA-subtype nerve agents exhibit high selectivity to proteins via lysine residues, which is critical for understanding the toxicity of GAs. In addition, we describe a strategy for the specific traceability of GA-related NA exposure in humans using two novel potential biomarkers.
2.1. Materials GA, MEGA, and EEGA were obtained with more than 97 % purity from the Laboratory of Analytical Chemistry, Research Institute of Chemical Defense (Beijing, China). The proteins used for in vitro experiments were procured from Sigma-Aldrich Ltd. Dithiothreitol (DTT), iodoacetamide (IAM), as well as the Spin Albumin/IgG Erasin Kit were purchased from Sangon Biotech Co. (Shanghai, China). The modified trypsin was sequencing grade and obtained from Roche Applied Science (Indianapolis, IN). The D-Tube™ Dialyzer Mini (MWCO 6−8 kDa, Novagen) and Amicon Ultra centrifugal filter devices (MWCO 10 kDa) were purchased from Millipore. The protein L 4 F F chromatography column (1 mL) were obtained from Yeasen Biotech Co., Ltd (Shanghai, China). All other chemicals and solvents (analytical grade) were purchased from Hanlonda Technology Development Co., Ltd (Beijing, China). 2.2. Exposure assays in vitro and in vivo A stock solution for individual nerve agents, having a concentration of 5 μg μL−1, was prepared in super-dry isopropanol for all assays and stored at 4 °C in dark. We designed exposure experiments for nerve agents with three methods: blood protein in vitro, plasma in vitro, and intravenous injection in vivo (Fig. 3a), and extraction of phosphonylated peptides after each exposure experiment (Fig. 3b). 2.2.1. Blood protein in vitro Proteins (albumin, immunoglobulin, and hemoglobin) (100 μL, 1 mg mL −1) in phosphate buffer (pH = 7.4) were incubated with a 100fold molar concentration excess of GAs solution for 20 h at 37 °C under shaken conditions. The samples were subsequently denatured with 8 M urea, reduced with 50 mM DTT solution, and sequentially alkylated with 0.5 M IAM. After being dialyzed against 10 mM ammonium bicarbonate (NH4HCO3), the samples were digested by addition of 20 μL of 20 μg mL−1 trypsin in 25 mM NH4HCO3 buffer at 37 °C for 12 h. 2.2.2. Plasma in vitro Pooled plasma (1 mL), diluted to 1/30 in phosphate buffer, was exposed to 100-fold GAs (100 μL, 5 μg μL −1). The sample was incubated at 37 °C for 16 h and shaken. The target proteins (i.e., albumin, IgG, or hemoglobin) for GAs-binding were purified from plasma for tryptic digestion. 2.2.3. Rabbit in vivo Animal research was conducted in accordance with the ethics committee of the Research Institute of Chemical Defense. Three normal rabbits (2 males and 1 female) with a bodyweight of 2.0 kg were injected intravenously into the ear vein at a dose of 0.5 × LD50: 60 μg GA in 0.2 mL of saline. 0.5 mL blood samples were collected at 6 h, 24 h, 48 h, 3 days, 5 days, 7 days, and 10 days after dosing and then centrifuged at 1000 g for 15 min to obtain rabbit plasma. The extracted GAs-binding target proteins were finished according to the plasma in vitro assay. The enzymatic peptide fractions were collected using Amicon Ultra centrifugal filter devices (MWCO 10 kDa) at 10,000 g for 10 min, and then lyophilized and analyzed by mass spectrometry.
2. Materials and methods Note: GA, MEGA, and EEGA are nerve agents, and are highly toxic (The median lethal doses (LD50) for GA, MEGA, and EEGA are 180 μg
2.3. Purification of target proteins Albumin was extracted from plasma (40 μL) by a Spin Albumin/IgG Erasin Kit and sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). In-gel enzymatic digestion was performed on the 65 kDa band (Shevchenko et al., 2006; Thiede et al., 2000) IgG was
Fig. 1. Chemical structures of GA, soman, and sarin. 2
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Fig. 2. The optimized geometry of GAs, (a) GA, (b) MEGA, and (c) EEGA.
bound active sites is essential for clarifying the binding mechanism for GAs to monitor nerve agent toxicity. Here, we selected human serum albumin (HSA, Gi: 113576) as a model blood protein of interest. GA, EEGA, and MEGA were used to reveal the GAs-HSA interactions. This is the first study examining the binding mechanism for GAs, and these results are summarized in Table 1. A class of phosphonylated active sites for HSA were identified, including 13 sites for GA, 14 sites for EEGA, and 12 sites for MEGA. Interestingly the three GAs, with different chemical structures, produce shared binding behavior on HAS; that is, they specifically phosphonylated lysine residues, rather than other amino acid residues. Serine and tyrosine, which are easily phosphonylated by other nerve agents, were not found to be modified. Thus, GAs are highly reactive to lysine residues, making lysine residues likely targets for phosphonylation. Comparing the active sites of GA, EEGA, and MEGA, we found that 10 HSA peptides were bound by all three GAs— K12, K190, K199, K212, K225, K351, K414, K432, K525, and K541. These 10 identified HSA peptides, all lysine residues, constitute a potential biomarker group for GAs on HSA. These findings support the development of a retrospective tool for tracing PeN and PCeN bond-based nerve agent exposure in humans. Peak area intensity for the modified HSA peptides was calculated to shed light on the reactivity. The results of this semi-quantitative analysis indicated that HSA reactivity was not only sensitive to the active sites, but also to the chemical structure of GAs. We observed that the reactivity for each GAs was similar-but not identical. The 10 co-modified HSA lysine residues had higher peak areas for the three GAs, while the other peptides had lower peak areas. GA was more reactive with K199 (2253.90), K541 (751.30), and K525 (306.78), while EEGA was more reactive with K541 (3193.91), K199 (2794.67), and K525 (698.67). Correspondingly, the most reactive sites are K525 (822.10), K199 (502.39), and K351 (395.82) for MEGA. K525 and K199 exhibited the highest reactivity to all three GAs compared to other HSA modified lysine residues, as shown in Fig. 4. Thus, K*QTALVELVK (K525) and LK*C*ASLQK (K199) on HSA are readily binding to GA-subtype NAs. This provides the basis for simple, “direct” approachesfor human biomonitoring and toxicological testing following prolonged exposure to a low dose of GAs.
purified using a protein L 4 F F chromatography column on AKTA prime purification system. 100 μg of purified IgG was treated according to the method in exposure assays in vitro. The crude hemoglobin was precipitated from red blood cells (10 μL) in 1 % HCl in acetone (v/v) and obtained after centrifugation and dialysis. The crude hemoglobin was further purified by size exclusion chromatography (sephadex LH-20 gel) to remove low molecular weight impurities. Then a molecular weight of 15,000–17,500 was determined by SDS-PAGE. The crude hemoglobin was sequentially digested with trypsin in a solution containing 50 mM NH4HCO3, 25 mM NaCl, and 100 mM DTT. 2.4. MS analysis and database search The enzymatic peptide fractions were analyzed by Q-orbitrap-MS coupled with nLC. The modified sites were searched using the Hisequest built-in Proteome Discoverer 2.1 (Thermo, USA). These experiments were carried out as described in our previous work (Fu et al., 2019, 2018; Sun et al., 2017, 2016). The approximate quantification for the modified peptides was determined using the following formula:
Peakareaintensity=
Areaofthemodifiedpeptide × 100,000 Areaofallpeptides
2.5. Molecular docking Induced-fit docking was performed through ligand placement (Triangle Matcher) as well as scoring (London dG), energy minimization (Amber10:EHT) and rescoring (GBVI/WSA dG) by the molecular operating environment (MOE v2018.0101)(2018)Molecular Operating Environment (MOE, 2018MOE v2018.0101)(2018) software. Three-dimensional structural model of human serum albumin (PDB code: 4PO0) was generated by deleting distant solvent, adding hydrogens, installing tethers, and calculating charges and performing initial refinement of the system based on Amber10: EHT force field. The 2D structures of tabun and etoposide were drawn in ChemBioDraw 2014 and converted to 3D structures in MOE through energy minimization. Cavity identification and labeled-residues were built by employing Discovery Studio (DS) 2018 (Accelrys Software Inc., San Diego, CA, U.S.). The pKa values of the ionizable residues and solvent accessible surface area (SASa) were calculated. MOE-Dock was employed to predict the binding mode and free binding energy.
3.2. Modification of rabbit IgG and human hemoglobin lysine residues by GAs
3. Results and discussion
To rule out the effect of protein profiles on the selectivity of binding sites, a comparative in vitro assay for GA on two other blood proteins, human hemoglobin (HHb, Gi: 122713) and rabbit IgG (Rb-IgG, Gi: 23683338), were performed. A total of 3 sites on HHb and 10 sites on Rb-IgG were phosphonylated, as shown in Table 2. Similarly, no other phosphonylated amino acid residues on the two proteins were
3.1. Human serum albumin active sites modified by GAs Nerve agents are more sensitive to amino acid residues with active side chains, such as serine, tyrosine, and lysine. Determination of GAs3
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Fig. 3. Summary of the experimental workflow.
consistent with our previous experimental results on albumin of other organismal (Sun et al., 2017). The identified peptides on different proteins can be used to track the traceability of GA-based exposure for other needs. For example, the three active peptides on HHb, with a 56days half-life, provide a longer detection window to identify GA-
identified, except for lysine residues. These results agree with the hypothesis that GA-subtype NAs have a certain selectivity to lysine residues under in vitro conditions independent of the substrate protein. To our knowledge, this is the first time characterizing the binding mechanism of GA and its derivatives to proteins. These findings are is 4
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Table 1 GAs-modified peptides on HSA. Binding sitesa
K4 K12 K41 K174 K190 K199 K212 K225 K281 K317 K351 K414 K444 K432 K475 K525 K541
Labeled peptides
DAHK*SEVAHR FK*DLGEENFK K*LVNEVT KAAFTECCQAADK* LDELRDEGK*ASSAK LK*C*ASLQK AFK*AWAVAR FPK*AEFAEVSK EC*CEK*PLLEK DVC*K*NYAEAK LAK*TYETTLEK K*VPQVSTPTLVEVSR HPEA*KR NLGK*VGSK VTK*C*C*TESLVNR K*QTALVELVK ATK*EQLK
Table 2 GA-modified peptides in human hemoglobin and rabbit IgG. Peak area intensity
Binding sites
GA
EEGA
MEGA
— 100.17 — — 457.04 2253.90 169.04 42.57 6.89 24.42 294.26 93.91 — 87.65 48.83 306.78 751.30
71.86 45.91 — 53.90 598.86 2794.67 219.58 115.78 — 49.90 0.19 39.92 7.39 479.09 — 698.67 3193.91
7.31 147.67 19.79 — 114.18 502.39 334.93 65.46 — — 395.82 274.03 — 243.58 — 822.10 167.46
K169 K172 K203 K277 K281 K293 K302 K341 K394 K12 K17 K100
Labeled peptides TNTK*VDK VDK*TVAPSTC* IFPPKPK*DTL C*K*VHNK K*ALPAPIEK TISK*AR GQPLEPK*VY NGK*AEDNYK HNHYTQK* VK*AAWGK AAWGK*VGAHAG VDPVNFK*LLSH
Gi number 23683338 23683338 23683338 23683338 23683338 23683338 23683338 23683338 23683338 122713 122713 122713
Peak area intensity
a
74.65 49.06 45.86 4.80 63.99 25.06 12.26 18.66 29.33 3.33 6.81 14.80
a The heavy chain constant region of IgG was selected for phosphonylation identification.
Table 3 GA-modified peptides in RSA in vitro and in vivo. Peptide
adducts after GAs poisoning. Monitoring phosphonylation of lysine residues on IgG is a potentially valuable approach to improve knowledge on chronic nerve agent poisoning symptoms, such as the decline in resistance after long-term low-dose exposure to organophosphorus pesticides (Grigoryan et al., 2008).
LVK*EVTDLAK K*EPERNEC*FLHHK YK*AILTEC*C*EAADK LDALK*EK EK*ALISAAQER C*ASIQK*FGDR AYK*AWALVR FPK*ADFTDISK IVTDLTK*VHK EC*C*DK*PILEK LGK*AYEATLK K*VPQVSTPTLVEISR LC*VLHEK*TPVSEK TPVSEK*VTK VTK*C*C*SESLVDR K*QTALVELVK
3.3. Modification of rabbit albumin in vivo by GAs To verify the binding mechanism and reactivity of these identified albumin active sites in complex physiological environments, we performed in vivo rabbit experiments investigating GA-based adduct formation with albumin. The results are shown in Table 3. The in vivo experimental data for GA binding to albumin agree with the in vitro experimental data. When exposed to GA, 16 sites in rabbit albumin (RSA) in vitro and 9 sites in the rabbit plasma in vitro were detected, whereas only 5 sites were detected in vivo in rabbit: K525, K414, K351, K188, and K186. These sites all involve in lysine residues, which agrees with the data for EEGA and MEGA to HSA. The 5 sites detected in vivo in rabbit had with higher peak area intensity than the in vitro experiment, revealing that the high reactivity peptides in vitro are prone to stabilize in vivo. Therefore, the highly reactive peptides for HSA from in vitro experiments can guide the
Residue
K44 K94 K162 K186 K188 K205 K212 K225 K240 K281 K351 K414 K466 K472 K475 K525
In vitro
In vivo
Albumin
Plasma
26.78 1.23 8.32 101.35 506.73 10.50 97.73 19.91 3.47 6.87 57.91 6.55 13.39 12.31 10.86 83.25
√
√ √ √
√ √
√
√ √
√ √
√ √
√
detection of human poisoning to GA-subtype NAs. We performed in vivo evaluation for metabolic stability of the modified sites by examining exposure time, as shown in Table 4. The 5 GA-labeled peptides in vivo persisted for different lengths of time. K186 was detected within 48 h, and K188 and K351 detection lasted for 5 days. K414 and K525 detection lasted for 7 days, an indicator of strong
Fig. 4. The reactivity of HSA lysine residues to GAs by peak area intensities. 5
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Table 4 Residual time of GA-lysine adducts in vivo with an injection dose of 0.5 x LD50. Peptides
LDALK*EKALISAAQER EK*ALISAAQER LGK*AYEATLK K*VPQVSTPTLVEISR K*QTALVELVK a
Residues
K186 K188 K351 K414 K525
Areaa
4.48 6.55 4.80 18.97 6.03
Time 6h
24 h
48 h
3d
5d
7d
10 d
√ √ √ √ √
√ √ √ √ √
√ √ √ √ √
— √ √ √ √
— √ √ √ √
— — — √ √
— — — — —
Area was represented by the peak area intensity of peptides at 6 h after injection of GA.
metabolic stability. By contrast, the amino acid sequences of HSA have a homology of 76.3 % with RSA (Gi: 44889024). Thus, the in vivo rabbit exposure experimental adducts better reflected phosphonylated adducts in humans. Among the 5 peptides in vivo, K*VPQVSTPTLVEISR (K351), LGK*AYEATLK (K414), and K*QTALVELVK (K525) were also located in the amino acid sequence of HSA. Importantly, these 3 HSA adducts were highly reactive to GAs during in vitro exposure. These results support the possibility that K351, K414, and K525 are potential biomarkers with high sensitivity, specificity and predictability in retrospective detection of human exposure to GAs. This is the first report on potential biomarkers of albumin for use in tracking GAs-poisoning in humans.
Table 5 Characteristic fragments of the GAs-lysine immonium. GAs
Phospho Lys immonium ion-NH3
Mass (amu)
Phospho Lys immonium ion
Mass (amu)
GA
219.1257
236.1522
MEGA
233.1413
250.1679
EEGA
247.1570
264.1835
3.4. MS/MS analysis of the GAs-albumin adducts Mass spectrometry has increasingly been applied to the identification of protein adducts, providing ample opportunities to evaluate binding sites (Bonichon et al., 2018; Dubrovskii et al., 2019; Seto et al., 2019). Obtaining accurate molecular mass is an important first step in adduct detection. The structural information for GAs-lysine adducts is illustrated in Fig. 5. After phosphonylation, the theoretical mass of lysine residues (128.0949) increase by the mass of the O- ethyl- N,Ndialkyl phosphonate group derived from the corresponding GAs, (i.e., 135.0449 for GA, 163.0762 for EEGA, and 149.0606 for MEGA). Therefore, the binding sites have been deduced from the mass changes of b-ions and y-ions. Phosphonylated lysine peptides are subjected to a sequence of cleavage reactions that yield characteristic fragment ions (Grigoryan et al., 2009). Therefore, except for the parent ions and the sequence band y-ions, the non-sequence characteristic fragments corresponding to a phosphonylated lysine immonium ion minus NH3 (Phospho Lys immonium ion-NH3) and phosphonylated lysine immonium ion (Phospho Lys immonium ion) were found in the MS/MS spectra. As shown in
Table 5, these characteristic ions provide a convenient way to screen peptides containing GAs-modified lysine residues. The representative MS/MS spectra of GAs-lysine adducts on albumin are shown in Fig. 6 and Fig. 7. All other MS/MS spectra of modified sites are included in Fig. S1- S13 of the Supplementary Material. The data shown in Fig. 6 are MS/MS spectra for the peptide K*QTALVELVK. The three spectra represent the adducts formed by GA, EEGA, and MEGA, respectively. The fragmentation of theoretical b and y ions is shown at the top of the figure. Mainly three kinds of important signals are marked, with blue lines for y-ion series, red lines for b-ion series, and green lines for parent ions and characteristic ions. The parent ions are doubly charged in the form of [M+2 H]2+ with an m/z of 632.3813 amu for GA and 646.3890 amu for EEGA and MEGA, which include the mass of the unmodified peptides plus an increased mass of O- ethyl- N,N-dialkyl phosphonate group of GAs. The major peaks in the green boxes are assigned to the characteristic fragments of lysinephosphonylated peptides, which identify that these peptides are labeled
Fig. 5. Mass changes during GAs-based lysine phosphonylation by. 6
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Fig. 6. MS/MS spectra of GAs-modified lysine residues in K*QTALVELVK peptide. (a) GA-K525. (b) EEGA-K525. (c) MEGA-K525.
series that extends from b2 to b8 fragments. Fig. 7 shows the MS/MS spectra of the K*VPQVSTPTLVEISR peptide, belonging to GA-K414 (a), EEGA-K414 (b), and MEGA-K414 (c). The labeled peptide sequence is verified by the y ion series from y1 to y10 and several b ion series. The b2 ion shows that the KV amino acid
on lysine. The y-series, continuing from y1 to y9, do not add any mass, suggesting that none of the residues in QTALVELVK show indication of being labeled. Thus, the C-terminal lysine is the only candidate site for phosphonylation in these albumin peptides. This evidence demonstrates that K525 is the modified site in the peptide, further confirmed by a b7
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Fig. 7. MS/MS spectra of GAs-modified lysine residues in the K*VPQVSTPTLVEISR peptide. (a) GA-K414. (b) EEGA-K414. (c) MEGA-K414.
labeled lysine residues as well as the interaction between GAs and proteins and their potential binding modes, we used computer-aided techniques to model the 10 key GAs-based lysine adducts on HSA. The crystal structure of HSA was selected as the template protein and was loaded into the MOE for structural analysis. The position of the 10 GAs-labeled HSA lysine residues and cavities were observed in the crystal structure of HSA using the Define Site tool in DS. These results are shown in Fig. 8. With the exception K225, all sites are located near the cavities of HSA, which are sensitive to binding
sequences on the peptide are phosphonylated by GAs. The proline residue has no active side chain to bind to NAs. Therefore, only K414 is a valid phosphonylation site. The major peaks in the green boxes, which are assigned to the characteristic fragments of lysine-phosphonylated peptides, clearly validate that these peptides are labeled on K414.
3.5. Structural analysis and molecular docking To better understand the structural and functional features of the 8
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SAS is greater than 15 % (Yuan et al., 2002). In this way, the 10 binding sites are classified as “exposed” or “buried”. The degree of exposure, reflecting the reactive activity of each site, was evaluated through this approach. We observe that the 10 GAs-labeled lysine residues were all in an "exposed" state. K351 and K225 have the largest %SAS value (63.116 % and 60.638 %, respectively) and, therefore, are most likely to collide with GAs. This calculation indicates that most of the identified lysine residues all easily collide with the ligand, corroborating the experimental studies regarding the activity of the modified sites to GAs on the one hand. To examine the interaction of the labeled sites with adjacent residues, we mapped out three types of interactions (i.e., hydrogen bond, electrostatic, and ion-pair) that significantly contributed to the site reactivity. The closer the residues are, the stronger their interaction. The results are shown in Table 7. Out of the 10 phosphonylated lysine residues, K199, K351, K414, and K525 formed two hydrogen bonds with corresponding residues. Among them, K525 had the strongest hydrogen bond (2.771), which increased the hydrophobicity of the K525-GAs adducts. K12, K190, K212, K432, and K541 formed one hydrogen bond. Meanwhile, K212 and K190 used the other types of interaction. Overall, the data suggest that K525 is the most significant lysine on HSA followed by K212 for tracing GAs toxicity in humans. To obtain detailed information about the interactions between labeled lysine residues and GA, we performed docking simulation studies. Redocking were conducted to evaluate the reliability of the established docking parameters. After re-docking the self-ligand of HAS (etoposide) to the protein, the root-mean-square distance was 0.35 Å, which indicated that the set-up parameters of molecular docking were reliable. In this approach, the 10 labeled lysine residues were selected as binding sites. The docking scores are shown in Fig. 9. The level of free binding energy was consistent with the covalent binding tendency obtained in the GAs exposure experiments. Moreover, K525 has the lowest binding energies (−5.18 kcal mol −1) of the 10 labeled lysine residues, closely followed by K199 (−5.13 kcal mol −1). Based on the minimum energy criterion, K525 is the most stable site for binding GA. These results are consistent with the experimental results obtained in section 3.1; that is, K525 and K199 are the most reactive lysine residues. In total, the data support the experimental result that K525 is the most reactive albumin residue to GAs. The binding mode of K525 was selected for interaction visualization. The 2D and 3D binding modes are shown in Fig. 10a and Fig. 10b, respectively. When GA is close to the HSA K525 site, the nitrogen atom of GA forms hydrogen bonds with the side chain of K525 and Y401. Meanwhile, The carbon atom of GA forms H-π conjugation with the side chain of F551. These two factors reduce the energy of the nucleophilic reaction between K525 and GA, making the GA-K525 adduct more stable and, theoretically, explaining the observed experimental phenomenon. Besides K525, the binding modes for the other nine sites are shown in Fig. S14 to Fig. S22 in the Supplementary Data.
Fig. 8. GA-labeled lysine residues position in the cavities of the HSA. Table 6 The predicted SASa of the 10 labeled lysine residues on HSA. Residues
SAS(Å2)
%SAS
Exposed/buried
pKa
K12 K190 K199 K212 K225 K351 K414 K432 K525 K541
107.578 109.466 45.424 60.192 117.515 117.921 29.547 29.285 64.931 122.740
55.320 56.290 23.358 30.953 60.430 60.638 15.194 15.059 33.389 63.116
Exposed Exposed Exposed Exposed Exposed Exposed Exposed Exposed Exposed Exposed
11.553 11.162 10.656 11.354 11.417 10.785 11.147 10.146 10.553 9.735
Table 7 The interaction of GAs-labeled lysine residues with other residues on HSA. Residues
Interaction
Interaction residue
Interaction type
K12
H-donor Positive H-donor Positive H-donor H-donor Positive H-donor Positive H-donor H-donor H-Acceptor H-donor H-donor H-donor H-donor H-Acceptor
A8 E57 R186 E425 K195 L203 E208 V216 E208 L347 T355 R472 L491 K436 R521 L529 K545
Hydrogen bond Electrostatic b Hydrogen bond Electrostatic Hydrogen bond Hydrogen bond Electrostatic Hydrogen bond Ion-pair Hydrogen bond Hydrogen bond Hydrogen bond Hydrogen bond Hydrogen bond Hydrogen bond Hydrogen bond Hydrogen bond
K190 K199 K212
K351 K414 K432 K525 K541
Distance (Å) a
3.217 3.095 2.934 3.908 3.289 3.075 2.477 2.949 2.480 2.879 3.134 3.225 2.805 2.943 3.404 2.771 2.942
4. Conclusions We developed a sensitive quadrupole-orbitrap mass spectrometry coupled with proteomics methodology approach for the identification of the active sites of GA-subtype nerve agents on the serum protein in vitro and in vivo samples. Preliminary data suggest that this method is sensitive, precise, and reliable enough to facilitate the discovery of new complementary biomarkers of nerve agents on high-abundance proteins, biological monitor and estimate the GA-subtype nerve agent exposure using the potential biomarkers screened above as a secondary tool in the field of forensic toxicology. It was revealed that all three of GAs—GA, MEGA, and EEGA expressed a robust selectivity for the lysine residues. A total of 10 GAslabeled HSA sites were identified in vitro, with K525 in K*QTALVELVK and K414 in K*VPQVSTPTLVEISR generated stable adducts in rabbit for 7 days after GA exposure. These results indicate that the two chemically
a
Hydrogen and electrostatic bonds formed within 3.5 Å identified. The electrostatic mainly referred to the ion-pair between basic amino acid residues and acidic amino acid residues within 3.5 Å. b
to GAs to satisfy the reaction requirements. To investigate whether the lysine residues were exposed for frequent collision with GAs, SASa for each residue was evaluated using the SASa in Å2 and relative SAS (%SAS). The value is listed in Table 6. We set a threshold for defining if a residue is solvent-accessible when the %
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Fig. 9. The free binding energy for the ten GAs-labeled HSA lysine residues.
Fig. 10. The binding mode between K525 and GA. (a) 2D schematic interaction diagram. (b) 3D binding mode. GA, yellow; the Interacting residues, green; and the backbone of HSA, light blue ribbon.
stable adducts are suitable as valuable forensic biomarkers, complementary to currently specific biomarkers, for the determination of the postmortem interval after poisoning. Specifically, future toxicokinetic studies of the most reactive peptide K*QTALVELVK on HSA in other animal models may confirm K525 as a promising tool for traceability and forensic analysis in terrorist attack victims, military, and research purposes after GA-related poisoning.
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Toxicology Letters journal homepage: www.elsevier.com/locate/toxlet
Protein adduct binding properties of tabun-subtype nerve agents after exposure in vitro and in vivo
T
Feiyan Fu, Haibo Liu, Runli Gao, Pengcheng Zhao, Xiaogang Lu, Ruihua Zhang, Liangliang Wang, Hongmei Wang*, Chengxin Pei* State Key Laboratory of NBC Protection for Civilian, Beijing 102205, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Nerve agents Tabun Albumin Q-Orbitrap Lysine adducts
Upon entering the body, nerve agents can bind active amino acid residues to form phosphonylated adducts. Tabun derivatives (O-alkyl-N,N-dialkyl phosphoroamidocyanidates) have strikingly different structural features from other G-series nerve agents, such as sarin and soman. Here, we investigate the binding mechanism for the phosphonylated adducts of nerve agents of tabun derivatives. Binding sites for three tabun derivatives, O-ethylN,N- dimethyl phosphoramidocyanidate (GA), O-ethyl-N,N-ethyl(methyl) phosphoramidocyanidate, and Oethyl-N,N-diethylphosphoramidocyanidate were studied. Quadrupole-orbitrap mass spectrometry (Q-OrbitrapMS) coupled to proteomics was used to screen adducts between tabun derivatives and albumin, immunoglobulin, and hemoglobin. The results reveal that all three tabun derivatives exhibit robust selectivity to lysine residues, rather than other amino acid residue types. A set of 10 lysine residues on human serum albumin are labeled by tabun derivatives in vitro, with K525 (K*QTALVELVK) and K199 (LK*CASLQK) peptides displaying the most reactivity. Tabun derivatives formed stable adducts on K525 and K414 (K*VPQVSTPTLVEVSR) for at least 7 days and on K351 (LAK*TYETTLEK) for at least 5 days in a rabbit model. Three of these peptides—K525, K414, and K351—have the highest homology with human serum albumin of all 5 lysine residues that bound to examined rabbit blood proteins in vivo. Molecular simulation of the tabun-albumin interaction using structural analysis and molecular docking provided theoretical evidence supporting lysine residue reactivity to phosphonylation by tabun derivatives. K525 has the lowest free binding energy and the strongest hydrogen bonding to human albumin. In summary, these findings identify unique binding properties for tabun derivatives to blood proteins.
1. Introduction Nerve agents (NAs) (Bajgar, 2004a) have been used since the World War II and are toxic and lethal chemical weapons that produce harmful effects on human health. After World War II, many hundred thousand tons of chemical warfare agents (CWA) were destroyed at sea. Among them, the coastal areas of Europe, Russia, Japan, and the United States have been affected by nerve agents, sulfur mustard, and Louise appearing to be the most common CWA disposed at sea. Since then, the international community has made it a priority to address the related environmental risks. Also, even if the use and acquisition of NAs is prohibited under the Chemical Weapon Convention (CWC) of 1993(1997)Chemical Weapon Convention (CWC) of, 2019Chemical Weapon Convention (CWC) of 1993(1997), NAs could be used by terrorists today that would pose a threat to human health and peace
(Bhaganagar and Bhimireddy, 2017; Nakagawa and Tu, 2018; Nepovimova and Kuca, 2018). Therefore, the search for better ways to quickly detect and monitor NAs has major importance for human safety and security, particularly for verification and traceable analysis in human poisoning events. The most commonly utilized method is the analysis of dialkyl phosphate metabolites (DAPs) and other metabolites in urine (Black et al., 1994; Shafik and Enos, 1969). However, these metabolites have half-lives ranging from 24 to 48 h, rendering this method futile for longterm detection of NAs in human poisoning events (Pardasani et al., 2007). Therefore, NA-protein adducts are potentially beneficial for tracing NA exposure since they have longer half-lives than DAPs and other metabolites related NA exposure (Black et al., 1999; Lee et al., 2018; Lockridge and Schopfer, 2010; Pardasani et al., 2007; Read et al., 2010; Schmidt et al., 2014; Schopfer and Lockridge, 2012).
⁎
Corresponding authors at: State Key Laboratory of NBC Protection for Civilian, Beijing, 102205, China. E-mail addresses: [email protected] (F. Fu), [email protected] (H. Liu), [email protected] (R. Gao), [email protected] (P. Zhao), [email protected] (X. Lu), [email protected] (R. Zhang), [email protected] (L. Wang), [email protected] (H. Wang), [email protected] (C. Pei). https://doi.org/10.1016/j.toxlet.2019.12.014 Received 2 August 2019; Received in revised form 9 December 2019; Accepted 13 December 2019 Available online 14 December 2019 0378-4274/ © 2019 Published by Elsevier B.V.
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kg−1, 400 μg kg -1, and 800 μg kg -1, respectively, i.v. in female mice). Personal protection is required against poisoning during all experiments.
Accordingly, a few proteins in blood plasma, such as cholinesterase, albumin, and hemoglobin, have become useful protein biomarkers for nerve agent exposure (Chen et al., 2013; Gupta et al., 2005; John et al., 2010; Peeples et al., 2005). The standard method used for biomarkers of nerve agent exposure is currently based on nine peptides on blood butyrylcholinesterase (BChE); however, the purification of NAs-ChE adducts is problematic. Albumin possesses advantages as a biomarker of exposure compared with the serine active on BChE. Tyrosine adducts are more stable and do not age like serine active sites on BChE. On the other hand, albumin present in much larger quantities compared with BChE. Further, albumin is present in much larger quantities compared with BChE. Thus, high abundant blood proteins may be more useful biomarkers compared with those from BchE. The G-series—which includes sarin (GB), soman (GD), and tabun (GA)—is one of the most important classes of nerve agents. O-ethylN,N- dimethyl phosphoramidocyanidate (GA) (Gupta et al., 1987; Heilbronn and Tolagen, 1965) belongs to the G-series nerve agents and produces acute toxicity on the nervous system through acetylcholinesterase (AChE) inhibition, quickly resulting in paralysis, seizure, and death (Bajgar, 2004b; Carletti et al., 2010; Mercey et al., 2012; Pardio et al., 2001; Schecter, 2004). In the 1980s, more than 400 people were poisoned in a chemical attack in the Iran-Iraq war, which has been investigated for the GA use (Haines and Fox, 2014). As a primary nerve agent, the structure of GA is more specific than the other NAs in the G-series family. The PeCN and PNe bonds in GA, which are different from the PeF and PCe bonds in sarin and soman (see Fig. 1), make the phosphonylation reactivity of GA with proteins distinctive. Covalent adducts are formed between nerve agents and proteins via phosphonylation—a nucleophilic substitution reaction. The existence of a lone electron pair located on the ortho-functional group (N,N-dialkyl) makes the nucleophilic attack of GA different from sarin and soman. On the other hand, since the leaving ability of CN- in GA is slightly weaker than the F- in sarin and soman, the nucleophilic reaction process for adduct formation is affected. Here, we examine the selectivity and phosphonylation of active sites on proteins by GA. We used three nerve agents (Fig. 2), GA, O-ethyl-N,N-ethyl(methyl) phosphoramidocyanidate (MEGA), and O-ethyl-N,N-diethylphosphoramidocyanidate (EEGA), which contain the same chemical structural framework, to represent GA derivatives (GAs). Three blood proteins—albumin, hemoglobin, and immunoglobulin (IgG) —were used to reveal the phosphonylation mechanism of GAs. To evaluate the binding selectivity of GAs to proteins, we performed exposure assays of GAs with proteins in vitro, plasma in vitro, and rabbit in vivo. The adducts bound by GAs were identified using quadrupole-Orbitrap mass spectrometry (Q-Orbitrap) coupled with nanoscale liquid chromatography method (nLC). Furthermore, molecular simulation studies examined the interaction between GA and albumin lysine residues. The results show that GA-subtype nerve agents exhibit high selectivity to proteins via lysine residues, which is critical for understanding the toxicity of GAs. In addition, we describe a strategy for the specific traceability of GA-related NA exposure in humans using two novel potential biomarkers.
2.1. Materials GA, MEGA, and EEGA were obtained with more than 97 % purity from the Laboratory of Analytical Chemistry, Research Institute of Chemical Defense (Beijing, China). The proteins used for in vitro experiments were procured from Sigma-Aldrich Ltd. Dithiothreitol (DTT), iodoacetamide (IAM), as well as the Spin Albumin/IgG Erasin Kit were purchased from Sangon Biotech Co. (Shanghai, China). The modified trypsin was sequencing grade and obtained from Roche Applied Science (Indianapolis, IN). The D-Tube™ Dialyzer Mini (MWCO 6−8 kDa, Novagen) and Amicon Ultra centrifugal filter devices (MWCO 10 kDa) were purchased from Millipore. The protein L 4 F F chromatography column (1 mL) were obtained from Yeasen Biotech Co., Ltd (Shanghai, China). All other chemicals and solvents (analytical grade) were purchased from Hanlonda Technology Development Co., Ltd (Beijing, China). 2.2. Exposure assays in vitro and in vivo A stock solution for individual nerve agents, having a concentration of 5 μg μL−1, was prepared in super-dry isopropanol for all assays and stored at 4 °C in dark. We designed exposure experiments for nerve agents with three methods: blood protein in vitro, plasma in vitro, and intravenous injection in vivo (Fig. 3a), and extraction of phosphonylated peptides after each exposure experiment (Fig. 3b). 2.2.1. Blood protein in vitro Proteins (albumin, immunoglobulin, and hemoglobin) (100 μL, 1 mg mL −1) in phosphate buffer (pH = 7.4) were incubated with a 100fold molar concentration excess of GAs solution for 20 h at 37 °C under shaken conditions. The samples were subsequently denatured with 8 M urea, reduced with 50 mM DTT solution, and sequentially alkylated with 0.5 M IAM. After being dialyzed against 10 mM ammonium bicarbonate (NH4HCO3), the samples were digested by addition of 20 μL of 20 μg mL−1 trypsin in 25 mM NH4HCO3 buffer at 37 °C for 12 h. 2.2.2. Plasma in vitro Pooled plasma (1 mL), diluted to 1/30 in phosphate buffer, was exposed to 100-fold GAs (100 μL, 5 μg μL −1). The sample was incubated at 37 °C for 16 h and shaken. The target proteins (i.e., albumin, IgG, or hemoglobin) for GAs-binding were purified from plasma for tryptic digestion. 2.2.3. Rabbit in vivo Animal research was conducted in accordance with the ethics committee of the Research Institute of Chemical Defense. Three normal rabbits (2 males and 1 female) with a bodyweight of 2.0 kg were injected intravenously into the ear vein at a dose of 0.5 × LD50: 60 μg GA in 0.2 mL of saline. 0.5 mL blood samples were collected at 6 h, 24 h, 48 h, 3 days, 5 days, 7 days, and 10 days after dosing and then centrifuged at 1000 g for 15 min to obtain rabbit plasma. The extracted GAs-binding target proteins were finished according to the plasma in vitro assay. The enzymatic peptide fractions were collected using Amicon Ultra centrifugal filter devices (MWCO 10 kDa) at 10,000 g for 10 min, and then lyophilized and analyzed by mass spectrometry.
2. Materials and methods Note: GA, MEGA, and EEGA are nerve agents, and are highly toxic (The median lethal doses (LD50) for GA, MEGA, and EEGA are 180 μg
2.3. Purification of target proteins Albumin was extracted from plasma (40 μL) by a Spin Albumin/IgG Erasin Kit and sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). In-gel enzymatic digestion was performed on the 65 kDa band (Shevchenko et al., 2006; Thiede et al., 2000) IgG was
Fig. 1. Chemical structures of GA, soman, and sarin. 2
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Fig. 2. The optimized geometry of GAs, (a) GA, (b) MEGA, and (c) EEGA.
bound active sites is essential for clarifying the binding mechanism for GAs to monitor nerve agent toxicity. Here, we selected human serum albumin (HSA, Gi: 113576) as a model blood protein of interest. GA, EEGA, and MEGA were used to reveal the GAs-HSA interactions. This is the first study examining the binding mechanism for GAs, and these results are summarized in Table 1. A class of phosphonylated active sites for HSA were identified, including 13 sites for GA, 14 sites for EEGA, and 12 sites for MEGA. Interestingly the three GAs, with different chemical structures, produce shared binding behavior on HAS; that is, they specifically phosphonylated lysine residues, rather than other amino acid residues. Serine and tyrosine, which are easily phosphonylated by other nerve agents, were not found to be modified. Thus, GAs are highly reactive to lysine residues, making lysine residues likely targets for phosphonylation. Comparing the active sites of GA, EEGA, and MEGA, we found that 10 HSA peptides were bound by all three GAs— K12, K190, K199, K212, K225, K351, K414, K432, K525, and K541. These 10 identified HSA peptides, all lysine residues, constitute a potential biomarker group for GAs on HSA. These findings support the development of a retrospective tool for tracing PeN and PCeN bond-based nerve agent exposure in humans. Peak area intensity for the modified HSA peptides was calculated to shed light on the reactivity. The results of this semi-quantitative analysis indicated that HSA reactivity was not only sensitive to the active sites, but also to the chemical structure of GAs. We observed that the reactivity for each GAs was similar-but not identical. The 10 co-modified HSA lysine residues had higher peak areas for the three GAs, while the other peptides had lower peak areas. GA was more reactive with K199 (2253.90), K541 (751.30), and K525 (306.78), while EEGA was more reactive with K541 (3193.91), K199 (2794.67), and K525 (698.67). Correspondingly, the most reactive sites are K525 (822.10), K199 (502.39), and K351 (395.82) for MEGA. K525 and K199 exhibited the highest reactivity to all three GAs compared to other HSA modified lysine residues, as shown in Fig. 4. Thus, K*QTALVELVK (K525) and LK*C*ASLQK (K199) on HSA are readily binding to GA-subtype NAs. This provides the basis for simple, “direct” approachesfor human biomonitoring and toxicological testing following prolonged exposure to a low dose of GAs.
purified using a protein L 4 F F chromatography column on AKTA prime purification system. 100 μg of purified IgG was treated according to the method in exposure assays in vitro. The crude hemoglobin was precipitated from red blood cells (10 μL) in 1 % HCl in acetone (v/v) and obtained after centrifugation and dialysis. The crude hemoglobin was further purified by size exclusion chromatography (sephadex LH-20 gel) to remove low molecular weight impurities. Then a molecular weight of 15,000–17,500 was determined by SDS-PAGE. The crude hemoglobin was sequentially digested with trypsin in a solution containing 50 mM NH4HCO3, 25 mM NaCl, and 100 mM DTT. 2.4. MS analysis and database search The enzymatic peptide fractions were analyzed by Q-orbitrap-MS coupled with nLC. The modified sites were searched using the Hisequest built-in Proteome Discoverer 2.1 (Thermo, USA). These experiments were carried out as described in our previous work (Fu et al., 2019, 2018; Sun et al., 2017, 2016). The approximate quantification for the modified peptides was determined using the following formula:
Peakareaintensity=
Areaofthemodifiedpeptide × 100,000 Areaofallpeptides
2.5. Molecular docking Induced-fit docking was performed through ligand placement (Triangle Matcher) as well as scoring (London dG), energy minimization (Amber10:EHT) and rescoring (GBVI/WSA dG) by the molecular operating environment (MOE v2018.0101)(2018)Molecular Operating Environment (MOE, 2018MOE v2018.0101)(2018) software. Three-dimensional structural model of human serum albumin (PDB code: 4PO0) was generated by deleting distant solvent, adding hydrogens, installing tethers, and calculating charges and performing initial refinement of the system based on Amber10: EHT force field. The 2D structures of tabun and etoposide were drawn in ChemBioDraw 2014 and converted to 3D structures in MOE through energy minimization. Cavity identification and labeled-residues were built by employing Discovery Studio (DS) 2018 (Accelrys Software Inc., San Diego, CA, U.S.). The pKa values of the ionizable residues and solvent accessible surface area (SASa) were calculated. MOE-Dock was employed to predict the binding mode and free binding energy.
3.2. Modification of rabbit IgG and human hemoglobin lysine residues by GAs
3. Results and discussion
To rule out the effect of protein profiles on the selectivity of binding sites, a comparative in vitro assay for GA on two other blood proteins, human hemoglobin (HHb, Gi: 122713) and rabbit IgG (Rb-IgG, Gi: 23683338), were performed. A total of 3 sites on HHb and 10 sites on Rb-IgG were phosphonylated, as shown in Table 2. Similarly, no other phosphonylated amino acid residues on the two proteins were
3.1. Human serum albumin active sites modified by GAs Nerve agents are more sensitive to amino acid residues with active side chains, such as serine, tyrosine, and lysine. Determination of GAs3
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Fig. 3. Summary of the experimental workflow.
consistent with our previous experimental results on albumin of other organismal (Sun et al., 2017). The identified peptides on different proteins can be used to track the traceability of GA-based exposure for other needs. For example, the three active peptides on HHb, with a 56days half-life, provide a longer detection window to identify GA-
identified, except for lysine residues. These results agree with the hypothesis that GA-subtype NAs have a certain selectivity to lysine residues under in vitro conditions independent of the substrate protein. To our knowledge, this is the first time characterizing the binding mechanism of GA and its derivatives to proteins. These findings are is 4
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Table 1 GAs-modified peptides on HSA. Binding sitesa
K4 K12 K41 K174 K190 K199 K212 K225 K281 K317 K351 K414 K444 K432 K475 K525 K541
Labeled peptides
DAHK*SEVAHR FK*DLGEENFK K*LVNEVT KAAFTECCQAADK* LDELRDEGK*ASSAK LK*C*ASLQK AFK*AWAVAR FPK*AEFAEVSK EC*CEK*PLLEK DVC*K*NYAEAK LAK*TYETTLEK K*VPQVSTPTLVEVSR HPEA*KR NLGK*VGSK VTK*C*C*TESLVNR K*QTALVELVK ATK*EQLK
Table 2 GA-modified peptides in human hemoglobin and rabbit IgG. Peak area intensity
Binding sites
GA
EEGA
MEGA
— 100.17 — — 457.04 2253.90 169.04 42.57 6.89 24.42 294.26 93.91 — 87.65 48.83 306.78 751.30
71.86 45.91 — 53.90 598.86 2794.67 219.58 115.78 — 49.90 0.19 39.92 7.39 479.09 — 698.67 3193.91
7.31 147.67 19.79 — 114.18 502.39 334.93 65.46 — — 395.82 274.03 — 243.58 — 822.10 167.46
K169 K172 K203 K277 K281 K293 K302 K341 K394 K12 K17 K100
Labeled peptides TNTK*VDK VDK*TVAPSTC* IFPPKPK*DTL C*K*VHNK K*ALPAPIEK TISK*AR GQPLEPK*VY NGK*AEDNYK HNHYTQK* VK*AAWGK AAWGK*VGAHAG VDPVNFK*LLSH
Gi number 23683338 23683338 23683338 23683338 23683338 23683338 23683338 23683338 23683338 122713 122713 122713
Peak area intensity
a
74.65 49.06 45.86 4.80 63.99 25.06 12.26 18.66 29.33 3.33 6.81 14.80
a The heavy chain constant region of IgG was selected for phosphonylation identification.
Table 3 GA-modified peptides in RSA in vitro and in vivo. Peptide
adducts after GAs poisoning. Monitoring phosphonylation of lysine residues on IgG is a potentially valuable approach to improve knowledge on chronic nerve agent poisoning symptoms, such as the decline in resistance after long-term low-dose exposure to organophosphorus pesticides (Grigoryan et al., 2008).
LVK*EVTDLAK K*EPERNEC*FLHHK YK*AILTEC*C*EAADK LDALK*EK EK*ALISAAQER C*ASIQK*FGDR AYK*AWALVR FPK*ADFTDISK IVTDLTK*VHK EC*C*DK*PILEK LGK*AYEATLK K*VPQVSTPTLVEISR LC*VLHEK*TPVSEK TPVSEK*VTK VTK*C*C*SESLVDR K*QTALVELVK
3.3. Modification of rabbit albumin in vivo by GAs To verify the binding mechanism and reactivity of these identified albumin active sites in complex physiological environments, we performed in vivo rabbit experiments investigating GA-based adduct formation with albumin. The results are shown in Table 3. The in vivo experimental data for GA binding to albumin agree with the in vitro experimental data. When exposed to GA, 16 sites in rabbit albumin (RSA) in vitro and 9 sites in the rabbit plasma in vitro were detected, whereas only 5 sites were detected in vivo in rabbit: K525, K414, K351, K188, and K186. These sites all involve in lysine residues, which agrees with the data for EEGA and MEGA to HSA. The 5 sites detected in vivo in rabbit had with higher peak area intensity than the in vitro experiment, revealing that the high reactivity peptides in vitro are prone to stabilize in vivo. Therefore, the highly reactive peptides for HSA from in vitro experiments can guide the
Residue
K44 K94 K162 K186 K188 K205 K212 K225 K240 K281 K351 K414 K466 K472 K475 K525
In vitro
In vivo
Albumin
Plasma
26.78 1.23 8.32 101.35 506.73 10.50 97.73 19.91 3.47 6.87 57.91 6.55 13.39 12.31 10.86 83.25
√
√ √ √
√ √
√
√ √
√ √
√ √
√
detection of human poisoning to GA-subtype NAs. We performed in vivo evaluation for metabolic stability of the modified sites by examining exposure time, as shown in Table 4. The 5 GA-labeled peptides in vivo persisted for different lengths of time. K186 was detected within 48 h, and K188 and K351 detection lasted for 5 days. K414 and K525 detection lasted for 7 days, an indicator of strong
Fig. 4. The reactivity of HSA lysine residues to GAs by peak area intensities. 5
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Table 4 Residual time of GA-lysine adducts in vivo with an injection dose of 0.5 x LD50. Peptides
LDALK*EKALISAAQER EK*ALISAAQER LGK*AYEATLK K*VPQVSTPTLVEISR K*QTALVELVK a
Residues
K186 K188 K351 K414 K525
Areaa
4.48 6.55 4.80 18.97 6.03
Time 6h
24 h
48 h
3d
5d
7d
10 d
√ √ √ √ √
√ √ √ √ √
√ √ √ √ √
— √ √ √ √
— √ √ √ √
— — — √ √
— — — — —
Area was represented by the peak area intensity of peptides at 6 h after injection of GA.
metabolic stability. By contrast, the amino acid sequences of HSA have a homology of 76.3 % with RSA (Gi: 44889024). Thus, the in vivo rabbit exposure experimental adducts better reflected phosphonylated adducts in humans. Among the 5 peptides in vivo, K*VPQVSTPTLVEISR (K351), LGK*AYEATLK (K414), and K*QTALVELVK (K525) were also located in the amino acid sequence of HSA. Importantly, these 3 HSA adducts were highly reactive to GAs during in vitro exposure. These results support the possibility that K351, K414, and K525 are potential biomarkers with high sensitivity, specificity and predictability in retrospective detection of human exposure to GAs. This is the first report on potential biomarkers of albumin for use in tracking GAs-poisoning in humans.
Table 5 Characteristic fragments of the GAs-lysine immonium. GAs
Phospho Lys immonium ion-NH3
Mass (amu)
Phospho Lys immonium ion
Mass (amu)
GA
219.1257
236.1522
MEGA
233.1413
250.1679
EEGA
247.1570
264.1835
3.4. MS/MS analysis of the GAs-albumin adducts Mass spectrometry has increasingly been applied to the identification of protein adducts, providing ample opportunities to evaluate binding sites (Bonichon et al., 2018; Dubrovskii et al., 2019; Seto et al., 2019). Obtaining accurate molecular mass is an important first step in adduct detection. The structural information for GAs-lysine adducts is illustrated in Fig. 5. After phosphonylation, the theoretical mass of lysine residues (128.0949) increase by the mass of the O- ethyl- N,Ndialkyl phosphonate group derived from the corresponding GAs, (i.e., 135.0449 for GA, 163.0762 for EEGA, and 149.0606 for MEGA). Therefore, the binding sites have been deduced from the mass changes of b-ions and y-ions. Phosphonylated lysine peptides are subjected to a sequence of cleavage reactions that yield characteristic fragment ions (Grigoryan et al., 2009). Therefore, except for the parent ions and the sequence band y-ions, the non-sequence characteristic fragments corresponding to a phosphonylated lysine immonium ion minus NH3 (Phospho Lys immonium ion-NH3) and phosphonylated lysine immonium ion (Phospho Lys immonium ion) were found in the MS/MS spectra. As shown in
Table 5, these characteristic ions provide a convenient way to screen peptides containing GAs-modified lysine residues. The representative MS/MS spectra of GAs-lysine adducts on albumin are shown in Fig. 6 and Fig. 7. All other MS/MS spectra of modified sites are included in Fig. S1- S13 of the Supplementary Material. The data shown in Fig. 6 are MS/MS spectra for the peptide K*QTALVELVK. The three spectra represent the adducts formed by GA, EEGA, and MEGA, respectively. The fragmentation of theoretical b and y ions is shown at the top of the figure. Mainly three kinds of important signals are marked, with blue lines for y-ion series, red lines for b-ion series, and green lines for parent ions and characteristic ions. The parent ions are doubly charged in the form of [M+2 H]2+ with an m/z of 632.3813 amu for GA and 646.3890 amu for EEGA and MEGA, which include the mass of the unmodified peptides plus an increased mass of O- ethyl- N,N-dialkyl phosphonate group of GAs. The major peaks in the green boxes are assigned to the characteristic fragments of lysinephosphonylated peptides, which identify that these peptides are labeled
Fig. 5. Mass changes during GAs-based lysine phosphonylation by. 6
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Fig. 6. MS/MS spectra of GAs-modified lysine residues in K*QTALVELVK peptide. (a) GA-K525. (b) EEGA-K525. (c) MEGA-K525.
series that extends from b2 to b8 fragments. Fig. 7 shows the MS/MS spectra of the K*VPQVSTPTLVEISR peptide, belonging to GA-K414 (a), EEGA-K414 (b), and MEGA-K414 (c). The labeled peptide sequence is verified by the y ion series from y1 to y10 and several b ion series. The b2 ion shows that the KV amino acid
on lysine. The y-series, continuing from y1 to y9, do not add any mass, suggesting that none of the residues in QTALVELVK show indication of being labeled. Thus, the C-terminal lysine is the only candidate site for phosphonylation in these albumin peptides. This evidence demonstrates that K525 is the modified site in the peptide, further confirmed by a b7
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Fig. 7. MS/MS spectra of GAs-modified lysine residues in the K*VPQVSTPTLVEISR peptide. (a) GA-K414. (b) EEGA-K414. (c) MEGA-K414.
labeled lysine residues as well as the interaction between GAs and proteins and their potential binding modes, we used computer-aided techniques to model the 10 key GAs-based lysine adducts on HSA. The crystal structure of HSA was selected as the template protein and was loaded into the MOE for structural analysis. The position of the 10 GAs-labeled HSA lysine residues and cavities were observed in the crystal structure of HSA using the Define Site tool in DS. These results are shown in Fig. 8. With the exception K225, all sites are located near the cavities of HSA, which are sensitive to binding
sequences on the peptide are phosphonylated by GAs. The proline residue has no active side chain to bind to NAs. Therefore, only K414 is a valid phosphonylation site. The major peaks in the green boxes, which are assigned to the characteristic fragments of lysine-phosphonylated peptides, clearly validate that these peptides are labeled on K414.
3.5. Structural analysis and molecular docking To better understand the structural and functional features of the 8
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SAS is greater than 15 % (Yuan et al., 2002). In this way, the 10 binding sites are classified as “exposed” or “buried”. The degree of exposure, reflecting the reactive activity of each site, was evaluated through this approach. We observe that the 10 GAs-labeled lysine residues were all in an "exposed" state. K351 and K225 have the largest %SAS value (63.116 % and 60.638 %, respectively) and, therefore, are most likely to collide with GAs. This calculation indicates that most of the identified lysine residues all easily collide with the ligand, corroborating the experimental studies regarding the activity of the modified sites to GAs on the one hand. To examine the interaction of the labeled sites with adjacent residues, we mapped out three types of interactions (i.e., hydrogen bond, electrostatic, and ion-pair) that significantly contributed to the site reactivity. The closer the residues are, the stronger their interaction. The results are shown in Table 7. Out of the 10 phosphonylated lysine residues, K199, K351, K414, and K525 formed two hydrogen bonds with corresponding residues. Among them, K525 had the strongest hydrogen bond (2.771), which increased the hydrophobicity of the K525-GAs adducts. K12, K190, K212, K432, and K541 formed one hydrogen bond. Meanwhile, K212 and K190 used the other types of interaction. Overall, the data suggest that K525 is the most significant lysine on HSA followed by K212 for tracing GAs toxicity in humans. To obtain detailed information about the interactions between labeled lysine residues and GA, we performed docking simulation studies. Redocking were conducted to evaluate the reliability of the established docking parameters. After re-docking the self-ligand of HAS (etoposide) to the protein, the root-mean-square distance was 0.35 Å, which indicated that the set-up parameters of molecular docking were reliable. In this approach, the 10 labeled lysine residues were selected as binding sites. The docking scores are shown in Fig. 9. The level of free binding energy was consistent with the covalent binding tendency obtained in the GAs exposure experiments. Moreover, K525 has the lowest binding energies (−5.18 kcal mol −1) of the 10 labeled lysine residues, closely followed by K199 (−5.13 kcal mol −1). Based on the minimum energy criterion, K525 is the most stable site for binding GA. These results are consistent with the experimental results obtained in section 3.1; that is, K525 and K199 are the most reactive lysine residues. In total, the data support the experimental result that K525 is the most reactive albumin residue to GAs. The binding mode of K525 was selected for interaction visualization. The 2D and 3D binding modes are shown in Fig. 10a and Fig. 10b, respectively. When GA is close to the HSA K525 site, the nitrogen atom of GA forms hydrogen bonds with the side chain of K525 and Y401. Meanwhile, The carbon atom of GA forms H-π conjugation with the side chain of F551. These two factors reduce the energy of the nucleophilic reaction between K525 and GA, making the GA-K525 adduct more stable and, theoretically, explaining the observed experimental phenomenon. Besides K525, the binding modes for the other nine sites are shown in Fig. S14 to Fig. S22 in the Supplementary Data.
Fig. 8. GA-labeled lysine residues position in the cavities of the HSA. Table 6 The predicted SASa of the 10 labeled lysine residues on HSA. Residues
SAS(Å2)
%SAS
Exposed/buried
pKa
K12 K190 K199 K212 K225 K351 K414 K432 K525 K541
107.578 109.466 45.424 60.192 117.515 117.921 29.547 29.285 64.931 122.740
55.320 56.290 23.358 30.953 60.430 60.638 15.194 15.059 33.389 63.116
Exposed Exposed Exposed Exposed Exposed Exposed Exposed Exposed Exposed Exposed
11.553 11.162 10.656 11.354 11.417 10.785 11.147 10.146 10.553 9.735
Table 7 The interaction of GAs-labeled lysine residues with other residues on HSA. Residues
Interaction
Interaction residue
Interaction type
K12
H-donor Positive H-donor Positive H-donor H-donor Positive H-donor Positive H-donor H-donor H-Acceptor H-donor H-donor H-donor H-donor H-Acceptor
A8 E57 R186 E425 K195 L203 E208 V216 E208 L347 T355 R472 L491 K436 R521 L529 K545
Hydrogen bond Electrostatic b Hydrogen bond Electrostatic Hydrogen bond Hydrogen bond Electrostatic Hydrogen bond Ion-pair Hydrogen bond Hydrogen bond Hydrogen bond Hydrogen bond Hydrogen bond Hydrogen bond Hydrogen bond Hydrogen bond
K190 K199 K212
K351 K414 K432 K525 K541
Distance (Å) a
3.217 3.095 2.934 3.908 3.289 3.075 2.477 2.949 2.480 2.879 3.134 3.225 2.805 2.943 3.404 2.771 2.942
4. Conclusions We developed a sensitive quadrupole-orbitrap mass spectrometry coupled with proteomics methodology approach for the identification of the active sites of GA-subtype nerve agents on the serum protein in vitro and in vivo samples. Preliminary data suggest that this method is sensitive, precise, and reliable enough to facilitate the discovery of new complementary biomarkers of nerve agents on high-abundance proteins, biological monitor and estimate the GA-subtype nerve agent exposure using the potential biomarkers screened above as a secondary tool in the field of forensic toxicology. It was revealed that all three of GAs—GA, MEGA, and EEGA expressed a robust selectivity for the lysine residues. A total of 10 GAslabeled HSA sites were identified in vitro, with K525 in K*QTALVELVK and K414 in K*VPQVSTPTLVEISR generated stable adducts in rabbit for 7 days after GA exposure. These results indicate that the two chemically
a
Hydrogen and electrostatic bonds formed within 3.5 Å identified. The electrostatic mainly referred to the ion-pair between basic amino acid residues and acidic amino acid residues within 3.5 Å. b
to GAs to satisfy the reaction requirements. To investigate whether the lysine residues were exposed for frequent collision with GAs, SASa for each residue was evaluated using the SASa in Å2 and relative SAS (%SAS). The value is listed in Table 6. We set a threshold for defining if a residue is solvent-accessible when the %
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Fig. 9. The free binding energy for the ten GAs-labeled HSA lysine residues.
Fig. 10. The binding mode between K525 and GA. (a) 2D schematic interaction diagram. (b) 3D binding mode. GA, yellow; the Interacting residues, green; and the backbone of HSA, light blue ribbon.
stable adducts are suitable as valuable forensic biomarkers, complementary to currently specific biomarkers, for the determination of the postmortem interval after poisoning. Specifically, future toxicokinetic studies of the most reactive peptide K*QTALVELVK on HSA in other animal models may confirm K525 as a promising tool for traceability and forensic analysis in terrorist attack victims, military, and research purposes after GA-related poisoning.
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Declaration of Competing Interest The authors declare that they have no conflict of interests. Acknowledgements This work was performed with the financial support of National Key R&D Program of China (No.2018YFC1602600). References (1997) Convention on the prohibition of the development, production, stockpiling and use of Chemical Weapons and their destruction, technical secretariat of the
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