Skip to main content
SpringerLink
Log in

A proteomics strategy for the identification of multiple sites in sulfur mustard–modified HSA and screening potential biomarkers for retrospective analysis of exposed human plasma

  • Research Paper
  • Published:
Analytical and Bioanalytical Chemistry Aims and scope Submit manuscript

Abstract

A major challenge for the unequivocal verification of alleged exposure to sulfur mustard (HD) lies in identifying its multiple modifications on endogenous proteins and utilizing these modified proteins to achieve accurate, sensitive, and rapid detection for retrospective analysis of HD exposure. As the most abundant protein in human plasma, human serum albumin (HSA) can react with many xenobiotics, such as HD, to protect the body from damage. The HSA adducts induced by HD have been used as biomarkers for the verification of HD exposure. In this study, the modification sites on HSA by HD were identified through application of the bottom-up strategy used in proteomics, and 41 modified sites were discovered with seven types of amino acids, of which 3 types were not previously reported. Then, different enzymes, including pepsin, endoproteinase Glu-C, and pronase, were applied to digest HD-HSA to produce adducts with hydroxyethylthioethyl (HETE) groups, which may be used as potential biomarkers for HD exposure. As candidates for retrospective analysis, sixteen adducts were obtained and characterized with ultra-high-pressure liquid chromatography coupled with quadrupole-Orbitrap mass spectrometry (UHPLC–QE Focus MS). These potential biomarkers were evaluated in human plasma that was exposed in vitro to HD and five of its analogues. This study integrated the identification of modification sites through application of the bottom-up strategy of proteomics and screening biomarkers, providing a novel strategy for retrospective detection of the exposure of xenobiotic chemicals.

Graphical abstract

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  1. Borak J, Sidell FR. Agents of chemical warfare: sulfur mustard. Ann Emerg Med. 1992;21(3):303–8.

    Article  CAS  PubMed  Google Scholar 

  2. Jan Y-H, Heck DE, Laskin DL, Laskin JD. DNA damage signaling in the cellular responses to mustard vesicants. Toxicol Lett. 2020;326:78–82.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Khan F, Niaz K, Hassan FI, Abdollahi M. An evidence-based review of the genotoxic and reproductive effects of sulfur mustard (vol 91, pg 1143, 2017). Arch Toxicol. 2018;92(7):2407–8.

    Article  CAS  PubMed  Google Scholar 

  4. Mishra N, Raina K, Agarwal R. Deciphering the role of microRNAs in mustard gas-induced toxicity. Ann NY Acad Sci. 2020.

  5. Ruszkiewicz JA, Buerkle A, Mangerich A. NAD(+) in sulfur mustard toxicity. Toxicol Lett. 2020;324:95–103.

    Article  CAS  PubMed  Google Scholar 

  6. Borna H, Noe SHHQ, Harchegani AB, Talatappe NR, Ghatrehsamani M, Ghanei M, et al. A review on proteomics analysis to reveal biological pathways and predictive proteins in sulfur mustard exposed patients: roles of inflammation and oxidative stress. Inhal Toxicol. 2019;31(1):3–11.

    Article  CAS  PubMed  Google Scholar 

  7. Lixin S, Lipeng PEI, Liming Z, Ju LI, Jian S. Effect of mustard gas at LD_(50) dose on reproductive system of rats. Acad J Second Mil Univ. 2007;28(6):637–9.

    Google Scholar 

  8. Azizi F, Keshavarz A, Roshanzamir F, Nafarabadi M. Reproductive function in men following exposure to chemical warfare with sulphur mustard. Med War. 1995;11(1):34–44.

    Article  CAS  PubMed  Google Scholar 

  9. Balali-Mood M, Hefazi M. The pharmacology, toxicology, and medical treatment of sulphur mustard poisoning. Fund Clin Pharmacol. 2005;19(3):297–315.

    Article  CAS  Google Scholar 

  10. Abbas F. Report of the specialists appointed by the Secretary-General of the United Nations to investigate allegations by the Islamic Republic of Iran concerning the use of chemical weapons. Mol Ecol. 1984;15(14):4577–88.

    Google Scholar 

  11. Prescott T. Accidental sulfur mustard exposure from explosive ordnance in a UK military service person. BMJ Mil Health. 2020.

  12. John H, Koller M, Worek F, Thiermann H, Siegert M. Forensic evidence of sulfur mustard exposure in real cases of human poisoning by detection of diverse albumin-derived protein adducts. Arch Toxicol. 2019;93(7):1881–91.

    Article  CAS  PubMed  Google Scholar 

  13. Xia Z. Chemical weapons: defense and destruction. Xia Z, editor. Beijing: Chemical Industry Publishing House; 2014.

  14. Orlova OI, Savel’eva EI, Khlebnikova NS. Methods for the detection of sulfur mustard metabolites in biological materials: an analytical review. J Anal Chem+. 2012;68(1):1–11.

  15. Popiel S, Nawala J, Dziedzic D, Soderstrom M, Vanninen P. Determination of mustard gas hydrolysis products thiodiglycol and thiodiglycol sulfoxide by gas chromatography-tandem mass spectrometry after trifluoroacetylation. Anal Chem. 2014;86(12):5865–72.

    Article  CAS  PubMed  Google Scholar 

  16. Halme M, Karjalainen M, Kiljunen H, Vanninen P. Development and validation of efficient stable isotope dilution LC-HESI-MS/MS method for the verification of beta-lyase metabolites in human urine after sulfur mustard exposure. J Chromatogr B. 2011;879(13–14):908–14.

    Article  CAS  Google Scholar 

  17. Bevan MJ, Wogen MT, Lunda MD, Saravia SA. High throughput quantitative analysis of the beta-lyase sulfur mustard metabolite, 1,1 ’-sulfonylbis 2-(methylsulfinyl)ethane in urine via high performance liquid chromatography tandem mass spectrometry. J Chromatogr B. 2017;1051:1–8.

    Article  CAS  Google Scholar 

  18. Liu CC, Liu SL, Xi HL, Yu HL, Zhou SK, Huang GL, et al. Simultaneous quantification of four metabolites of sulfur mustard in urine samples by ultra-high performance liquid chromatography-tandem mass spectrometry after solid phase extraction. J Chromatogr A. 2017;1492:41–8.

    Article  CAS  PubMed  Google Scholar 

  19. Yue LJ, Wei YX, Chen J, Shi HQ, Liu Q, Zhang YJ, et al. Abundance of four sulfur mustard-DNA adducts ex vivo and in vivo revealed by simultaneous quantification in stable isotope dilution-ultrahigh performance liquid chromatography-tandem mass spectrometry. Chem Res Toxicol. 2014;27(4):490–500.

    Article  CAS  PubMed  Google Scholar 

  20. Zhang YJ, Yue LJ, Nie ZY, Chen J, Guo L, Wu BD, et al. Simultaneous determination of four sulfur mustard-DNA adducts in rabbit urine after dermal exposure by isotope-dilution liquid chromatography-tandem mass spectrometry. J Chromatogr B. 2014;961:29–35.

    Article  CAS  Google Scholar 

  21. Black RM, Clarke RJ, Harrison JM, Read RW. Biological fate of sulphur mustard: identification of valine and histidine adducts in haemoglobin from casualties of sulphur mustard poisoning. Xenobiotica. 1997;27(5):499–512.

    Article  CAS  PubMed  Google Scholar 

  22. Noort D, Fidder A, Hulst AG, Woolfitt AR, Ash D, Barr JR. Retrospective detection of exposure to sulfur mustard: improvements on an assay for liquid chromatography-tandem mass spectrometry analysis of albumin/sulfur mustard adducts. J Anal Toxicol. 2004;28(5):333–8.

    Article  CAS  PubMed  Google Scholar 

  23. Steinritz D, Striepling E, Rudolf K-D, Schroeder-Kraft C, Pueschel K, Hullard-Pulstinger A, et al. Medical documentation, bioanalytical evidence of an accidental human exposure to sulfur mustard and general therapy recommendations. Toxicol Lett. 2016;244:112–20.

    Article  CAS  PubMed  Google Scholar 

  24. Steinritz D, Lueling R, Siegert M, Herbert J, Mueckter H, Taeger CD, et al. Alkylated epidermal creatine kinase as a biomarker for sulfur mustard exposure: comparison to adducts of albumin and DNA in an in vivo rat study. Arch Toxicol. 2021.

  25. Schmeisser W, Lueling R, Steinritz D, Thiermann H, Rein T, John H. Transthyretin as a target of alkylation and a potential biomarker for sulfur mustard poisoning: electrophoretic and mass spectrometric identification and characterization. Drug Test Anal. 2021.

  26. Lueling R, Singer H, Popp T, John H, Boekhoff I, Thiermann H, et al. Sulfur mustard alkylates steroid hormones and impacts hormone function in vitro. Arch Toxicol. 2019;93(11):3141–52.

    Article  CAS  Google Scholar 

  27. Grigoryan H, Edmands W, Lu SS, Yano Y, Regazzoni L, Iavarone AT, et al. Adductomics pipeline for untargeted analysis of modifications to Cys34 of human serum albumin. Anal Chem. 2016;88(21):10504–12.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Andacht TM, Pantazides BG, Crow BS, Fidder A, Noort D, Thomas JD, et al. Enhanced throughput method for quantification of sulfur mustard adducts to human serum albumin via isotope dilution tandem mass spectrometry. J Anal Toxicol. 2014;38(1):8–15.

    Article  CAS  PubMed  Google Scholar 

  29. Liu C, Liang L, Xiang Y, Yu H, Zhou S, Xi H, et al. An improved method for retrospective quantification of sulfur mustard exposure by detection of its albumin adduct using ultra-high pressure liquid chromatography-tandem mass spectrometry. Anal Bioanal Chem. 2015;407(23):7037–46.

    Article  CAS  PubMed  Google Scholar 

  30. Pantazides BG, Quinones-Gonzalez J, Nazario DMR, Crow BS, Perez JW, Blake TA, et al. A quantitative method to detect human exposure to sulfur and nitrogen mustards via protein adducts. J Chromatogr B. 2019;1121:9–17.

    Article  CAS  Google Scholar 

  31. Gandor F, Gawlik M, Thiermann H, John H. Evidence of sulfur mustard exposure in human plasma by LC-ESI-MS-MS detection of the albumin-derived alkylated HETE-CP dipeptide and chromatographic investigation of its Cis/Trans isomerism. J Anal Toxicol. 2015;39(4):270–9.

    Article  CAS  PubMed  Google Scholar 

  32. John H, Willoh S, Hoermann P, Siegert M, Vondran A, Thiermann H. Procedures for analysis of dried plasma using microsampling devices to detect sulfur mustard-albumin adducts for verification of poisoning. Anal Chem. 2016;88(17):8787–94.

    Article  CAS  PubMed  Google Scholar 

  33. Chen B, Yu H-L, Liu S-L, Liu C-C, Liang L-H, Li X-H, et al. A sensitive quantification approach for detection of HETE-CP adduct after benzyl chloroformate derivatization using ultra-high-pressure liquid chromatography tandem mass spectrometry. Anal Bioanal Chem. 2019;411(15):3405–15.

    Article  CAS  PubMed  Google Scholar 

  34. Richter A, Siegert M, Thiermann H, John H. Alkylated albumin-derived dipeptide C(-HETE)P derivatized by propionic anhydride as a biomarker for the verification of poisoning with sulfur mustard. Anal Bioanal Chem. 2021;413(19):4907–16.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. John H, Richter A, Thiermann H. Evidence of sulfur mustard poisoning by detection of the albumin-derived dipeptide biomarker C(-HETE)P after nicotinylation. Drug Test Anal. 2021.

  36. John H, Siegert M, Gandor F, Gawlik M, Kranawetvogl A, Karaghiosoff K, et al. Optimized verification method for detection of an albumin-sulfur mustard adduct at Cys(34) using a hybrid quadrupole time-of-flight tandem mass spectrometer after direct plasma proteolysis. Toxicol Lett. 2016;244:103–11.

    Article  CAS  PubMed  Google Scholar 

  37. Siegert M, Gandor F, Kranawetvogl A, Boerner H, Thiermann H, John H. Methionine(329) in human serum albumin: a novel target for alkylation by sulfur mustard. Drug Test Anal. 2019;11(5):659–68.

    Article  CAS  PubMed  Google Scholar 

  38. Work Instruction for the Reporting of the Results of the OPCW Biomedical Proficiency Tests. Technical Secretariat of OPCW. 2021.

  39. Final Report of Sixth Official OPCW Biomedical Proficiency Test. Technical Secretariat of OPCW. 2021.

  40. Yang Y, Shu Y-Z, Humphreys WG. Label-free bottom-up proteomic workflow for simultaneously assessing the target specificity of covalent drug candidates and their off-target reactivity to selected proteins. Chem Res Toxicol. 2016;29(1):109–16.

    Article  CAS  PubMed  Google Scholar 

  41. Fu F, Gao R, Zhang R, Zhao P, Lu X, Li L, et al. Verification of soman-related nerve agents via detection of phosphonylated adducts from rabbit albumin in vitro and in vivo. Arch Toxicol. 2019;93(7):1853–63.

    Article  CAS  PubMed  Google Scholar 

  42. Bollineni RC, Hoffmann R, Fedorova M. Identification of protein carbonylation sites by two-dimensional liquid chromatography in combination with MALDI- and ESI-MS. J Proteomics. 2011;74(11):2338–50.

    Article  CAS  PubMed  Google Scholar 

  43. Zhao MZ, Wu FL, Xu P. Development of a rapid high-efficiency scalable process for acetylated Sus scrofa cationic trypsin production from Escherichia coli inclusion bodies. Protein Expres Purif. 2015;116:120–6.

    Article  CAS  Google Scholar 

  44. Bent HE. THE LABORATORY PREPARATION OF MUSTARD GAS. Science. 1947;106(2755):374–5.

    Article  CAS  PubMed  Google Scholar 

  45. Williams AH, Woodward FN. New organic sulphur vesicants; analogues of 2:2'-dichlorodiethyl sulphide and 2:2'-di(chloroethylthio)diethyl ether. ether. J Chem Soc. 1948;16:38–42.

  46. Cox J, Mann M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat Biotechnol. 2008;26(12):1367–1372.

  47. Wisniewski JR, Zougman A, Nagaraj N, Mann M. Universal sample preparation method for proteome analysis. Nat Methods. 2009;6(5):359–62.

    Article  CAS  PubMed  Google Scholar 

  48. Smith JR, Capacio BR, Korte WD, Woolfitt AR, Barr JR. Analysis for plasma protein biomarkers following an accidental human exposure to sulfur mustard. J Anal Toxicol. 2008;32(1):17–24.

    Article  CAS  PubMed  Google Scholar 

  49. Black RM, Harrison JM, Read RW. Biological fate of sulphur mustard: in vitro alkylation of human haemoglobin by sulphur mustard. Xenobiotica. 1997;27(1):11–32.

    Article  CAS  PubMed  Google Scholar 

  50. Lawrence RJ, Smith JR, Boyd BL, Capacio BR. Improvements in the methodology of monitoring sulfur mustard exposure by gas chromatography-mass spectrometry analysis of cleaved and derivatized blood protein adducts. J Anal Toxicol. 2008;32(1):31–6.

    Article  CAS  PubMed  Google Scholar 

  51. Young SA, Capacio BR. Gas chromatography-tandem mass spectrometry verification of sulfur mustard exposure in humans through the conversion of protein adducts to free sulfur mustard. Chem Res Toxicol. 2020;33(7):1941–9.

    Article  CAS  PubMed  Google Scholar 

  52. Chen B, Yu H, Liu S, Liu C, Liang L, Yang Y, et al. Analysis of the sulfur mustard adduct to human hemoglobin in blood samples exposed to trace sulfur mustard by UHPLC-MS/MS. J Chin Mass Spec Soc. 2019;40(4):305–13.

    CAS  Google Scholar 

  53. Timperley CM, Black RM, Bird M, Holden I, Mundy JL, Read RW. Hydrolysis and oxidation products of the chemical warfare agents 1,2-bis (2-chloroethyl)thio ethane Q and 2,2 ’-bis(2-chloroethylthio)diethyl ether T. Phosphorus Sulfur Silicon. 2003;178(9):2027–46.

    Article  CAS  Google Scholar 

  54. Interim design assessment for the blue grass chemical agent destruction pilot plan. In: Council NR, editor. Washington, DC: The National Academies Press; 2005.

Download references

Funding

This work was supported by the State Key Laboratory of NBC Protection for Civilian (Grant No. SKLNBC2019-12), the Innovation Foundation of Medicine (AWS17J008), and the CAMS Innovation Fund for Medical Sciences (2019RU006).

Author information

Authors and Affiliations

  1. State Key Laboratory of NBC Protection for Civilian, Laboratory of Analytical Chemistry, Research Institute of Chemical Defence, Beijing, 102205, People’s Republic of China

    Bo Chen, Qiaoli Zhang, Huilan Yu, Changcai Liu, Yang Yang & Shilei Liu

  2. State Key Laboratory of Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences (Beijing), Research Unit of Proteomics & Research, Development of New Drug of Chinese Academy of Medical Sciences, Institute of Lifeomics, Beijing, 102206, People’s Republic of China

    Tao Zhang & Ping Xu

  3. School of Chemistry and Chemical Engineering, Nanjing University of Sciences & Technology, Nanjing, 210094, People’s Republic of China

    Zhe Ren

Authors
  1. Bo Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Qiaoli Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Zhe Ren
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Tao Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Huilan Yu
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Changcai Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Yang Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Ping Xu
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Shilei Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Ping Xu or Shilei Liu.

Ethics declarations

Ethics approval

The studies with human plasma were performed under the general ethical guidelines (Ethical review of biomedical research involving humans) on conducting scientific research which was approved and disseminated by the National Health Commission of the People’s Republic of China, 2016.

Informed consent

All written informed consent was obtained from all participants whose plasma samples were used.

Conflict of interest

The authors declare no competing interests.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (DOCX 4353 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Chen, B., Zhang, Q., Ren, Z. et al. A proteomics strategy for the identification of multiple sites in sulfur mustard–modified HSA and screening potential biomarkers for retrospective analysis of exposed human plasma. Anal Bioanal Chem 414, 4179–4188 (2022). https://doi.org/10.1007/s00216-022-04070-y

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00216-022-04070-y

Keywords

Search

Navigation