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Proteomic identification and quantification of Clostridium perfringens enterotoxin using a stable isotope-labelled peptide via liquid chromatography–tandem mass spectrometry

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Abstract

Purpose

Detection of Clostridium perfringens enterotoxin (CPE) in human stool is critical evidence of food poisoning. However, processing patient-derived samples is difficult and very few methods exist to confirm the presence of CPE. In this study, a technique was developed using proteomic analysis to identify and quantify CPE in artificial gut fluid as an alternative.

Methods

The standard CPE was spiked into artificial gut fluids, and effective methods were developed by employing both a stable isotope-labelled internal standard peptide and liquid chromatography–tandem mass spectrometry (LC–MS/MS).

Results

Proteotypic peptide EILDLAAATER formed by tryptic digestion was selected for quantitation of CPE. The peptide was identified using product ion spectra. Although the nontoxic peptides originating from CPE showed very low detectability in extraction and tryptic digestion, they could be detected with sufficient sensitivity using the method we developed. Based on a spiked recovery test at two concentrations (50 and 200 µg/kg), the recovery values were 85 and 78%, respectively. The relative standard deviations of repeatability and within-laboratory reproducibility were less than 8 and 11%, respectively. These standard deviations satisfied the criteria of the Japanese validation guidelines for residues (MHLW 2010, Director Notice, Syoku-An No. 1224–1). The limit of quantification (LOQ) was estimated to be 50 µg/kg. The combination of the product ion spectra and relative ion ratio supported CPE identification at the LOQ level.

Conclusions

To the best of our knowledge, this is the first report of proteomic analysis of CPE using LC–MS/MS. The method would greatly help in assessing CPE reliably.

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Data availability statement

The datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request.

References

  1. Duracova M, Klimentova J, Myslivcova Fucikova A, Zidkova L, Sheshko V, Rehulkova H, Dresler J, Krocova Z (2019) Targeted mass spectrometry analysis of Clostridium perfringens toxins. Toxins 11:177. https://doi.org/10.3390/toxins11030177

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Uzal FA, Freedman JC, Shrestha A, Theoret JR, Garcia J, Awad MM, Adams V, Moore RJ, Rood JI, McClane BA (2014) Towards an understanding of the role of Clostridiums perfringens toxins in human and animal disease. Future Microbiol 9:361–377. https://doi.org/10.2217/fmb.13.168

    Article  CAS  PubMed  Google Scholar 

  3. Duracova M, Klimentova J, Fucikova A, Dresler J (2018) Proteomic methods of detection and quantification of protein toxins. Toxins 10:99. https://doi.org/10.3390/toxins10030099

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Freedman JC, Shrestha A, McClane BA (2016) Clostridium perfringens enterotoxin: action, genetics, and translational applications. Toxins (Basel) 8:73. https://doi.org/10.3390/toxins8030073

    Article  CAS  PubMed  Google Scholar 

  5. National Institute of Infectious Diseases (NIID), Tokyo, Japan (2012) Manual for the Detection of Pathogenic Microorganisms. https://www.niid.go.jp/niid/ja/labo-manual.html. Accessed 11 Aug 2021.

  6. Berry PR, Rodhouse JC, Hughes S, Bartholomew BA, Gilbert RJ (1988) Evaluation of ELISA, RPLA, and vero cell assays for detecting clostridium perfringens enterotoxin in Faecal specimens. J Clin Pathol 41:458–461. https://doi.org/10.1136/jcp.41.4.458

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Mahony DE, Gilliatt E, Dawson S, Stockdale E, Lee SH (1983) Vevo cell assay for rapid detection of Clostridium perfringens enterotoxin. Appl Environ Microbiol 55:2141–2143

    Article  Google Scholar 

  8. Fisher DJ, Miyamoto K, Harrison B, Akimoto S, Sarker MR, McClane BA (2005) Association of beta2 toxin production with clostridium perfringens type A human gastrointestinal disease isolates carrying a plasmid enterotoxin gene. Mol Microbiol 56:747–762. https://doi.org/10.1111/j.1365-2958.2005.04573.x

    Article  CAS  PubMed  Google Scholar 

  9. Ishioka T, Aihara Y, Carle Y, Shigemura H, Kubomura A, Motoya T, Nakamoto A, Nakamura A, Fujimoto S, Hirai S, Oishi K, Nagaoka H, Kimura H, Murakami K (2020) Contrasting results from two commercial kits testing for the presence of Clostridium perfringens Enterotoxin in feces from norovirus-infected human patients. Clin Lab 66:929–936. https://doi.org/10.7754/Clin.Lab.2019.190801

    Article  CAS  Google Scholar 

  10. Lukinmaa S, Takkunen E, Siitonen A (2002) Molecular epidemiology of clostridium perfringens related to food-borne outbreaks of disease in Finland from 1984 to 1999. Appl Environ Microbiol 68:3744–3749. https://doi.org/10.1128/AEM.68.8.3744-3749.2002

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Gilquin B, Jaquinod M, Louwagie M, Kieffer-Jaquinod S, Kraut A, Ferro M, Becher F, Brun V (2017) A proteomics assay to detect eight CBRN-relevant toxins in food. Proteomics 17:1–2. https://doi.org/10.1002/pmic.201600357

    Article  CAS  Google Scholar 

  12. Marx V (2013) Targeted proteomics. Nat Methods 10:19–22. https://doi.org/10.1038/nmeth.2285

    Article  CAS  PubMed  Google Scholar 

  13. Kamiie J, Aihara N, Shirota K (2016) Basics and practices of quantitative proteomics using selected reaction monitoring (SRM). Proteome Lett 1:57–62

    Google Scholar 

  14. The Ministry of Education, Culture, Sports, Science, and Technology (MECSST). The Ministry of Health, Labour and Welfare (MHLW), and The Ministry of Economy, Trade and Industry (METI), Tokyo, Japan (2013) Ethical guidelines for human genome and genetic analysis research. Notification No. 1

  15. Ministry of Health, Labour and Welfare (MHLW), Tokyo, Japan (1996) Director notice, Ei-Ka No. 29

  16. Food Safety Commission of Japan (2004) Standards for safety assessments of food additives produced using genetically modified microorganisms. Commission Decision of 25 March 2004.

  17. Chen L, Li X, Pang Y, Li L, Zhang X, Yu L (2007) Resistant starch as a carrier for oral colon-targeting drug matrix system. J Mater Sci Mater Med 18:2199–2203. https://doi.org/10.1007/s10856-007-3009-6

    Article  CAS  PubMed  Google Scholar 

  18. Marques MRC, Loebenberg R, Almukainzi M (2011) Simulated biological fluids with possible application in dissolution testing. Dissolution Technol 18:15–28. https://doi.org/10.14227/DT180311P15

    Article  CAS  Google Scholar 

  19. Trailović JN, Stefanović S, Trailović SM (2013) In vitro and in vivo protective effects of three mycotoxin adsorbents against ochratoxin A in broiler chickens. Br Poult Sci 54:515–523. https://doi.org/10.1080/00071668.2013.798627

    Article  CAS  PubMed  Google Scholar 

  20. Kawakami H, Kamiie J, Yasuno K, Kobayashi R, Aihara N, Shirota K (2012) Dynamics of absolute amount of nephrin in a single podocyte in puromycin aminonucleoside nephrosis rats calculated by quantitative glomerular proteomics approach with selected reaction monitoring mode. Nephrol Dial Transplant 27:1324–1330. https://doi.org/10.1093/ndt/gfr492

    Article  CAS  PubMed  Google Scholar 

  21. Kaji H, Kamiie J, Kawakami H, Kido K, Yamauchi Y, Shinkawa T, Taoka M, Takahashi N, Isobe T (2007) Proteomics reveals N-linked glycoprotein diversity in Caenorhabditis elegans and suggests an atypical translocation mechanism for integral membrane proteins. Mol Cell Proteomics 6:2100–2109. https://doi.org/10.1074/mcp.M600392-MCP200

    Article  CAS  PubMed  Google Scholar 

  22. Ministry of Health, Labour and Welfare (MHLW), Tokyo, Japan (2010) Director notice; guideline for the validation of analytical methods for agricultural chemical residues in food. Syoku-An No. 1224–1

  23. Eurachem, Guide CITAC (2012) Quantifying uncertainty in analytical measurement, 3rd edn. https://www.eurachem.org/index.php/publications/guides/quam

  24. Navarro MA, McClane BA, Uzal FA (2018) Mechanisms of action and cell death associated with Clostridium perfringens Toxins. Toxins (Basel) 10:212. https://doi.org/10.3390/toxins10050212

    Article  CAS  PubMed  Google Scholar 

  25. Ministry of Health, Labour, and Welfare (MHLW), Tokyo, Japan (2020) Food poisoning statistics (data for foodborne diseases outbreaks). https://www.mhlw.go.jp/toukei/list/112-1.html. Accessed 11 Aug 2021.

  26. Picotti P, Lam H, Campbell D, Deutsch EW, Mirzaei H, Ranish J, Domon B, Aebersold R (2008) A database of mass spectrometric assays for the yeast proteome. Nat Methods 5:913–914. https://doi.org/10.1038/nmeth1108-913

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Maiolica A, Jünger MA, Ezkurdia I, Aebersold R (2012) Targeted proteome investigation via selected reaction monitoring mass spectrometry. J Proteomics 75:3495–3513. https://doi.org/10.1016/j.jprot.2012.04.048

    Article  CAS  PubMed  Google Scholar 

  28. Narumi R, Shimizu Y, Ueda HR (2017) Protein absolute quantification using cell-free-synthesized peptide. Proteome Lett 2:75–82

    Google Scholar 

  29. Yocum AK, Chinnaiyan AM (2009) Current affairs in quantitative targeted proteomics: Multiple reaction monitoring-mass spectrometry. Brief Funct Genomic Proteomic 8:145–157. https://doi.org/10.1093/bfgp/eln056

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Lynch KL, Breaud AR, Vandenberghe H, Wu AH, Clarke W (2010) Performance evaluation of three liquid chromatography mass spectrometry methods for broad spectrum drug screening. Clin Chim Acta 411:1474–1481. https://doi.org/10.1016/j.cca.2010.05.046

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Xing Y, Meng W, Sun W, Li D, Yu Z, Tong L, Zhao Y (2016) Simultaneous qualitative and quantitative analysis of 21 mycotoxins in Radix Paeoniae Alba by ultra-high performance liquid chromatography quadrupole linear ion trap mass spectrometry and QuEChERS for sample preparation. J Chromatogr B 1031:202–213. https://doi.org/10.1016/j.jchromb.2016.07.008

    Article  CAS  Google Scholar 

  32. Zawahir S, Gawarammana I, Dargan PI, Abdulghni M, Dawson AH (2017) Activated charcoal significantly reduces the amount of colchicine released from Gloriosa superba in simulated gastric and intestinal media. Clin Toxicol (Phila) 55:914–918. https://doi.org/10.1080/15563650.2017.1325897

    Article  CAS  PubMed  Google Scholar 

  33. Yamagaki T, Kimura Y, Yamazaki T (2021) Practical protocol for optimization of suppression of peptide adsorption in the sensitive quantitative LC-MS using calibration curve parameters. J Mass Spectrom Soc Jpn 69:23–29. https://doi.org/10.5702/massspec.20-115

    Article  CAS  Google Scholar 

  34. SANTE/12682/2019 (2020) Guidance document on analytical quality control and method validation procedures for pesticide residues and analysis in food and feed. (Document No. SANTE/12682/2019)

  35. Ippoushi K, Sasanuma M, Oike H, Kobori M, Maeda-Yamamoto M (2015) Absolute quantification of protein NP24 in tomato fruit by liquid chromatography/tandem mass spectrometry using stable isotope-labelled tryptic peptide standard. Food Chem 173:238–242. https://doi.org/10.1016/j.foodchem.2014.10.008

    Article  CAS  PubMed  Google Scholar 

  36. Montowska M, Fornal E (2019) Absolute quantification of targeted meat and allergenic protein additive peptide markers in meat products. Food Chem 274:857–864. https://doi.org/10.1016/j.foodchem.2018.08.131

    Article  CAS  PubMed  Google Scholar 

  37. Gabig TG, Waltzer WC, Whyard T, Romanov V (2016) Clostridium perfringens enterotoxin as a potential drug for intravesical treatment of bladder cancer. Biochem Biophys Res Commun 478:887–892. https://doi.org/10.1016/j.bbrc.2016.08.046

    Article  CAS  PubMed  Google Scholar 

  38. Nakashima C, Yamamoto K, Kishi S, Sasaki T, Ohmori H, Fujiwara-Tani R, Mori S, Kawahara I, Nishiguchi Y, Mori T, Kondoh M, Luo Y, Kirita T, Kuniyasu H (2020) Clostridium perfringens enterotoxin induces claudin-4 to activate YAP in oral squamous cell carcinomas. Oncotarget 11:309–321. https://doi.org/10.18632/oncotarget.27424

    Article  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

The authors would like to thank Jun’ichi Nakajima from the Department of Pharmaceutical and Environmental Science at the Tokyo Metropolitan Institute of Public Health for his helpful review of this manuscript.

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Authors and Affiliations

  1. Department of Food Safety, Tokyo Metropolitan Institute of Public Health, 3-24-1, Hyakunin-Cho, Shinjuku-Ku, Tokyo, 169-0073, Japan

    Hiroshi Koike, Maki Kanda, Souichi Yoshikawa, Hiroshi Hayashi, Yoko Matsushima, Yumi Ohba, Momoka Hayashi, Chieko Nagano, Kenji Otsuka & Takeo Sasamoto

  2. Laboratory of Veterinary Pathology, School of Veterinary Medicine, Azabu University, 1-17-71, Fuchinobe, Chuo-Ku, Sagamihara, Kanagawa, 252-5201, Japan

    Junichi Kamiie

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  1. Hiroshi Koike
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Contributions

Conceptualization: HK; investigation: HK; validation: HK; writing—original draft: HK; writing—review and editing: MK; project administration: MK; data curation: SY, HH, YM, YO, MH, and CN; resources: SY and HH; supervision: KO, JK and TS; methodology: JK.

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Correspondence to Hiroshi Koike.

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The authors declare that they have no known competing financial interests or personal relationships that could have influenced the work reported in this paper.

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Supplementary Information

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11419_2023_660_MOESM1_ESM.tif

Supplementary file1 Comparison of peak shapes in LC-MS/MS chromatograms using organic mobile phases: (a) acetonitrile containing 0.1% formic acid and (b) methanol containing 0.1% formic acid. Each peptide was obtained in Clostridium perfringens enterotoxin (CPE) standard solution at 1000 µg/L: (left) the quantitative transition (m/z 601.3/618.3), and (right) the transition (m/z 596.8/591.3). (TIF 155 KB)

11419_2023_660_MOESM2_ESM.tif

Supplementary file2 Comparison of background signals in total ion current chromatograms of blank artificial gut fluid samples (m/z 60–660) (a) without and (b) with the desalting process (GL-Tip SDB). The black arrow indicates the ion suppression. (TIF 67 KB)

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Koike, H., Kanda, M., Yoshikawa, S. et al. Proteomic identification and quantification of Clostridium perfringens enterotoxin using a stable isotope-labelled peptide via liquid chromatography–tandem mass spectrometry. Forensic Toxicol 41, 249–259 (2023). https://doi.org/10.1007/s11419-023-00660-2

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