Advertisement

Journal of The American Society for Mass Spectrometry

, Volume 29, Issue 9, pp 1812–1825 | Cite as

Structural Characterization and Absolute Quantification of Microcystin Peptides Using Collision-Induced and Ultraviolet Photo-Dissociation Tandem Mass Spectrometry

  • Troy J. Attard
  • Melissa D. Carter
  • Mengxuan Fang
  • Rudolph C. Johnson
  • Gavin E. Reid
Focus: Application of Photons and Radicals for MS: Research Article

Abstract

Microcystin (MC) peptides produced by cyanobacteria pose a hepatotoxic threat to human health upon ingestion from contaminated drinking water. While rapid MC identification and quantification in contaminated body fluids or tissue samples is important for patient treatment and outcomes, conventional immunoassay-based measurement strategies typically lack the specificity required for unambiguous determination of specific MC variants, whose toxicity can significantly vary depending on their structures. Furthermore, the unambiguous identification and accurate quantitation of MC variants using tandem mass spectrometry (MS/MS)-based methods can be limited due to a current lack of appropriate stable isotope-labeled internal standards. To address these limitations, we have systematically examined here the sequence and charge state dependence to the formation and absolute abundance of both “global” and “variant-specific” product ions from representative MC-LR, MC-YR, MC-RR, and MC-LA peptides, using higher-energy collisional dissociation (HCD)-MS/MS, ion-trap collision-induced dissociation (CID)-MS/MS and CID-MS3, and 193 nm ultraviolet photodissociation (UPVD)-MS/MS. HCD-MS/MS was found to provide the greatest detection sensitivity for both global and variant-specific product ions in each of the MC variants, except for MC-YR where a variant-specific product uniquely formed via UPVD-MS/MS was observed with the greatest absolute abundance. A simple methodology for the preparation and characterization of 18O-stable isotope-labeled MC reference materials for use as internal standards was also developed. Finally, we have demonstrated the applicability of the methods developed herein for absolute quantification of MC-LR present in human urine samples, using capillary scale liquid chromatography coupled with ultra-high resolution / accurate mass spectrometry and HCD-MS/MS.

Graphical abstract

Keywords

Microcystin Tandem mass spectrometry Ultraviolet photodissociation Absolute quantitation 

Notes

Disclaimer

The findings and conclusions in this study are those of the authors and do not necessarily represent the views of the US Department of Health and Human Services or the US Centers for Disease Control and Prevention. Use of trade names and commercial sources is for identification only and does not constitute endorsement by the US Department of Health and Human Services or the US Centers for Disease Control and Prevention.

Funding Information

Financial support for this work was received under contract 200-2014-59255 from The Centers for Disease Control and Prevention (CDC), National Center for Environmental Health (NCEH), Division of Laboratory Sciences (DLS), Emergency Response Branch (ERB), Atlanta, Georgia, USA.

Supplementary material

13361_2018_1981_MOESM1_ESM.docx (390 kb)
ESM 1 (DOCX 390 kb)

References

  1. 1.
    Zurawell, R.W., Chen, H., Burke, J.M., Prepas, E.E.: Hepatotoxic cyanobacteria: a review of the biological importance of microcystins in freshwater environments. J. Toxicol. Environ. Health B Crit. Rev. 8, 1–37 (2005)CrossRefGoogle Scholar
  2. 2.
    Vasconcelos, J.F., Barbosa, J.E.L., Lira, W., Azevedo, S.M.F.O.: Microcystin bioaccumulation can cause potential mutagenic effects in farm fish. Egypt. J. Aquat. Res. 39, 185–192 (2013)CrossRefGoogle Scholar
  3. 3.
    Pavagadhia, S., Balasubramanian, R.: Toxicological evaluation of microcystins in aquatic fish species: current knowledge and future directions. Aquat. Toxicol. 142–143, 1–16 (2013)CrossRefGoogle Scholar
  4. 4.
    DeVries, S.E., Galey, F.D., Namikoshi, M., Woo, J.C.: Clinical and pathologic findings of blue-green algae (Microcystis aeruginosa) intoxication in a dog. J. Vet. Diagn. Investig. 5, 403–408 (1993)CrossRefGoogle Scholar
  5. 5.
    Briand, J.-F., Jacquet, S., Bernard, C., Humbert, J.-F.: Health hazards for terrestrial vertebrates from toxic cyanobacteria in surface water ecosystems. Vet. Res. 34, 361–377 (2003)CrossRefGoogle Scholar
  6. 6.
    Gilroy, D.J., Kauffman, K.W., Hall, R.A., Huang, X., Chu, F.S.: Assessing potential health risks from microcystin toxins in blue-green algae dietary supplements. Environ. Health Perspect. 108, 435–439 (2000)CrossRefGoogle Scholar
  7. 7.
    Vichi, S., Lavorini, P., Funari, E., Scardala, S., Testai, E.: Contamination by Microcystis and microcystins of blue-green algae food supplements (BGAS) on the Italian market and possible risk for the exposed population. Food Chem. Toxicol. 50, 4493–4499 (2012)CrossRefGoogle Scholar
  8. 8.
    Herranz, S., Bocková, M., Marazuela, M.D., Homola, J., Moreno-Bondi, M.C.: An SPR biosensor for the detection of microcystins in drinking water. Anal. Bioanal. Chem. 398, 2625–2634 (2010)CrossRefGoogle Scholar
  9. 9.
    Cook, D., Newcombe, G.: Comparison and modeling of the adsorption of two microcystin analogues onto powdered activated carbon. Environ. Technol. 29, 525–534 (2008)CrossRefGoogle Scholar
  10. 10.
    Yu, S.-Z.: Primary prevention of hepatocellular carcinoma. J. Gastroenterol. Hepatol. 10, 674–682 (1995)CrossRefGoogle Scholar
  11. 11.
    Ueno, Y., Nagata, S., Tsutsumi, T., Hasegawa, A., Watanabe, M.-F., Park, H.-D., Chen, G.-C., Chen, G., Yus, S.-Z.: Detection of microcystins, a blue-green algal hepatotoxin, in drinking water sampled in Haimen and Fusui, endemic areas of primary liver cancer in China, by highly sensitive immunoassay. Carcinogenesis. 17, 1317–1321 (1996)CrossRefGoogle Scholar
  12. 12.
    Svirčev, Z., Krstić, S., Miladinov-Mikov, M., Baltić, V., Vidović, M.: Freshwater cyanobacterial blooms and primary liver cancer epidemiological studies in Serbia. J. Environ. Sci. Health Part C. 27, 36–55 (2009)CrossRefGoogle Scholar
  13. 13.
    Diehnelt, C.W., Peterman, S.M., Budde, W.L.: Liquid chromatography–tandem mass spectrometry and accurate m/z measurements of cyclic peptide cyanobacteria toxins. Trends Anal. Chem. 24, 622–634 (2005)Google Scholar
  14. 14.
    Khreich, N., Lamourette, P., Renard, P.-Y., Clavé, G., Fenaille, F., Créminon, C., Volland, H.: A highly sensitive competitive enzyme immunoassay of broad specificity quantifying microcystins and nodularins in water samples. Toxicon. 53, 551–559 (2009)CrossRefGoogle Scholar
  15. 15.
    de Silva, E.D., Williams, D.E., Andersen, R.J.: Motuporin, a potent protein phosphatase inhibitor isolated from the Papua New Guinea sponge Theonella swinhoei Gray. Tet. Lett. 33, 1561–1564 (1992)CrossRefGoogle Scholar
  16. 16.
    Hotto, A.M., Satchwell, M.F., Berry, D.L., Gobler, C.J., Boyer, G.L.: Spatial and temporal diversity of microcystins and microcystin-producing genotypes in Oneida Lake, NY. Harmful Algae. 7, 671–681 (2008)CrossRefGoogle Scholar
  17. 17.
    Namikoshi, M., Rinehart, K.L., Sakai, R.: Identification of 12 hepatotoxins from a Homer Lake bloom of the cyanobacteria Microcystis aeruginosa, Microcystis viridis, and Microcystis wesenbergii: nine new microcystins. J. Org. Chem. 57, 866–872 (1992)CrossRefGoogle Scholar
  18. 18.
    Namikoshi, M., Rinehart, K.L., Sakai, R.: Structures of three new cyclic heptapeptide hepatotoxins produced by the cyanobacterium (blue-green alga) Nostoc sp. strain 152. J. Org. Chem. 55, 6135–6139, 1990Google Scholar
  19. 19.
    Harada, K., Ogawa, K., Matsuura, K., Murata, H., Suzuki, M.: Structural determination of geometrical isomers of microcystins LR and RR from cyanobacteria by two-dimensional NMR spectroscopic techniques. Chem. Res. Toxicol. 3, 473–481 (1990)CrossRefGoogle Scholar
  20. 20.
    Puerto, M., Pichardo, S., Jos, A., Camean, A.M.: Comparison of the toxicity induced by microcystin-RR and microcystin-YR in differentiated and undifferentiated Caco-2 cells. Toxicon. 54, 161–169 (2009)CrossRefGoogle Scholar
  21. 21.
    Namikoshi, M., Choi, B.W., Sun, F., Rinehart, K.L., Evans, W.R., Carmichael, W.W.: Chemical characterization and toxicity of dihydro derivatives of nodularin and microcystin-LR, potent cyanobacterial cyclic peptide hepatotoxins. Chem. Res. Toxicol. 6, 151–158 (1993)CrossRefGoogle Scholar
  22. 22.
    Namikoshi, M., Sun, F., Choi, B.W., Rinehart, K.L., Carmichael, W.W., Evans, W.R.: Seven more microcystins from Homer Lake cells: application of the general method for structure assignment of peptides containing alpha-, beta-dehydroamino acid unit(s). J. Org. Chem. 60, 3671–3679 (1995)CrossRefGoogle Scholar
  23. 23.
    Harada, K., Ogawa, K., Kimura, Y., Murata, H., Suzuki, M., Thorn, P.M., Evans, W.R., Carmichael, W.W.: Microcystins from Anabaena flos-aquae NRC 525-17. Chem. Res. Toxicol. 4, 535–540 (1991)CrossRefGoogle Scholar
  24. 24.
    Stotts, R.R., Namikoshi, M., Haschek, W.M., Rinehart, K.L., Carmichael, W.W., Dahlem, A.M., Beasley, V.R.: Structural modifications imparting reduced toxicity in microcystins from Microcystis spp. Toxicon. 31, 783–789 (1993)CrossRefGoogle Scholar
  25. 25.
    Sherlock, I.R., James, K.J., Caudwell, F.B., MacKintosh, C.: First identification of microcystins in Irish lakes aided by a new derivatisation procedure for electrospray mass spectrometric analysis. Nat. Toxins. 5, 247–254 (1997)CrossRefGoogle Scholar
  26. 26.
    Geis-Asteggiante, L., Lehotay, S.J., Fortis, L.L., Paoli, G., Wijey, C., Heinzen, H.: Development and validation of a rapid method for microcystins in fish and comparing LC-MS/MS results with ELISA. Anal. Bioanal. Chem. 401, 2617–2630 (2011)CrossRefGoogle Scholar
  27. 27.
    Yuan, M., Namikoshi, M., Otsuki, A., Rinehart, K.L., Sivonen, K., Watanabe, M.F.: Low-energy collisionally activated decomposition and structural characterization of cyclic heptapeptide microcystins by electrospray ionization mass spectrometry. J. Mass Spectrom. 34, 33–34 (1999)CrossRefGoogle Scholar
  28. 28.
    Mayumi, T., Kato, H., Imanishi, S., Kawasaki, Y., Hasegawa, M., Harada, K.: Structural characterization of microcystins by LC/MS/MS under ion trap conditions. J. Antibiot. 59, 710–719 (2006)CrossRefGoogle Scholar
  29. 29.
    Frias, H.V., Mendes, M.A., Cardozo, K.H.M., Carvalho, V.M., Tomazela, D., Colepicolo, P., Pinto, E.: Use of electrospray tandem mass spectrometry for identification of microcystins during a cyanobacterial bloom event. Biochem. Bioph. Res. Co. 344, 741–746 (2006)CrossRefGoogle Scholar
  30. 30.
    de Graaf, E.L., Altelaar, A.F.M., van Breukelen, B., Mohammed, S., Heck, A.J.R.: Improving SRM assay development: a global comparison between triple quadrupole, ion trap, and higher energy CID peptide fragmentation spectra. J. Proteome Res. 10, 4334–4341 (2011)CrossRefGoogle Scholar
  31. 31.
    Brodbelt, J.S.: Ion activation methods for peptides and proteins. Anal. Chem. 88, 30–51 (2016)CrossRefGoogle Scholar
  32. 32.
    Brodbelt, J.S.: Photodissociation mass spectrometry: new tools for characterization of biological molecules. Chem. Soc. Rev. 43, 2757–2783 (2014)CrossRefGoogle Scholar
  33. 33.
    Theisen, A., Yan, B., Brown, J.M., Morris, M., Bellina, B., Barran, P.E.: Use of ultraviolet photodissociation coupled with ion mobility mass spectrometry to determine structure and sequence from drift time selected peptides and proteins. Anal. Chem. 88, 9964–9971 (2016)CrossRefGoogle Scholar
  34. 34.
    Madsen, J.A., Boutz, D.R., Brodbelt, J.S.: Ultrafast ultraviolet photodissociation at 193 nm and its applicability to proteomic workflows. J. Prot. Res. 9, 4205–4214 (2010)CrossRefGoogle Scholar
  35. 35.
    Fort, K.L., Dyachenko, A., Potel, C.M., Corradini, E., Marino, F., Barendregt, A., Makarov, A.A., Scheltema, R.A., Heck, A.J.R.: Implementation of ultraviolet photodissociation on a benchtop Q exactive mass spectrometer and its application to phosphoproteomics. Anal. Chem. 88, 2303–2310 (2016)CrossRefGoogle Scholar
  36. 36.
    Cleland, T.P., DeHart, C.J., Fellers, R.T., VanNispen, A.J., Greer, J.B., LeDuc, R.D., Parker, W.R., Thomas, P.M., Kelleher, N.L., Brodbelt, J.S.: High-throughput analysis of intact human proteins using UVPD and HCD on an Orbitrap mass spectrometer. J. Prot. Res. 16, 2072–2079 (2017)CrossRefGoogle Scholar
  37. 37.
    O'Brien, J.P., Li, W., Zhang, Y., Brodbelt, J.S.: Characterization of native protein complexes using ultraviolet photodissociation mass spectrometry. J. Am. Chem. Soc. 136, 12920–12908 (2014)CrossRefGoogle Scholar
  38. 38.
    Mistarz, U.H., Bellina, B., Jensen, P.F., Brown, J.M., Barran, P.E., Rand, K.D.: UV Photodissociation mass spectrometry accurately localize sites of backbone deuteration in peptides. Anal. Chem. 90, 1077–1080 (2018)CrossRefGoogle Scholar
  39. 39.
    Crittenden, C.M., Parker, W.R., Jenner, Z.B., Bruns, K.A., Akin, L.D., McGee, W.M., Ciccimaro, E., Brodbelt, J.S.: Exploitation of the ornithine effect enhances characterization of stapled and cyclic peptides. J. Am. Soc. Mass Spectrom. 27(5), 856–863 (2016)CrossRefGoogle Scholar
  40. 40.
    Ozawa, K.; Fujioka, H.; Muranaka, M.; Yokoyama, A.; Katagami, Y.; Homma, T.; Ishikawa, K.; Tsujimura, S.; Kumagai, M.; Watanabe, M.F.; Park, H-D. Spatial distribution and temporal variation of Microcystis species composition and microcystin concentration in Lake Biwa. Environ. Toxicol. 20, 270–276 (2005)Google Scholar
  41. 41.
    Szlag, D.C., Sinclair, J.L., Southwell, B., Westrick, J.A.: Cyanobacteria and cyanotoxins occurrence and removal from five high-risk conventional treatment drinking water plants. Toxins. 7, 2198–2220 (2015)CrossRefGoogle Scholar
  42. 42.
    Flores, C., Caixach, J.: An integrated strategy for rapid and accurate determination of free and cell-bound microcystins and related peptides in natural blooms by liquid chromatography-electrospray-high resolution mass spectrometry and matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry using both positive and negative ionization modes. J. Chromatogr. A. 1407, 76–89 (2015)CrossRefGoogle Scholar
  43. 43.
    Ryan, E., Nguyen, C.Q.N., Shiea, C., Reid, G.E.: Detailed structural characterization of sphingolipids via 193 nm ultraviolet Photodissociation and ultra high resolution tandem mass spectrometry. J. Am. Soc. Mass Spectrom. 28, 1406–1419 (2017)CrossRefGoogle Scholar
  44. 44.
    Miles, C.O., Sandvik, M., Nonga, H.E., Rundberget, T., Wilkins, A.L., Rise, F., Ballot, F.: Identification of microcystins in a Lake Victoria cyanobacterial bloom using LC-MS with thiol derivatization. Toxicon. 70, 21–31 (2013)CrossRefGoogle Scholar
  45. 45.
    Boyd, R., Somogy, Á.: The mobile proton hypothesis in fragmentation of protonated peptides: a perspective. J. Am. Soc. Mass Spectrom. 21, 1275–1278 (2010)CrossRefGoogle Scholar
  46. 46.
    Grach-Pogrebinsky, O., Sedmak, B., Carmeli, S.: Seco[D-Asp3]microcystin-RR and [D-Asp3,D-Glu(OMe)6]microcystin-RR, two new microcystins from a toxic water bloom of the cyanobacterium planktothrixrubescens. J. Nat. Prod. 67, 337–342 (2004)CrossRefGoogle Scholar
  47. 47.
    Brittain, S., Mohamed, S.A., Wang, J., Lehmann, V.K.B., Carmichael, W.W., Rinehart, K.L.: Isolation and characterization of microcystins from a River Nile strain of Oscillatoria tenuis Agardh ex Gomont. Toxicon. 38, 1759–1771 (2000)CrossRefGoogle Scholar
  48. 48.
    Pearson, C., Rinehart, K.L., Sugano, M., Costerison, J.R.: Enantiospecific synthesis of N-BOC-ADDA: a linear approach. Org. Lett. 2, 2901–2903 (2000)CrossRefGoogle Scholar
  49. 49.
    Clavé, G., Ronco, C., Boutal, H., Kreich, N., Volland, H., Franck, X., Romieu, A., Renard, P.-Y.: Facile and rapid access to linear and truncated microcystin analogues for the implementation of immunoassays. Org. Biomol. Chem. 8, 676–690 (2010)CrossRefGoogle Scholar
  50. 50.
    Naidu, B.N., Sorenson, M.E., Connolly, T.P., Ueda, Y.: Michael addition of amines and thiols to dehydroalanine amides: a remarkable rate acceleration in water. J. Org. Chem. 68, 10098–10102 (2003)CrossRefGoogle Scholar
  51. 51.
    Haaf, E., Schlosser, A.: Peptide and protein quantitation by acid-catalyzed 18O-labeling of carboxyl groups. Anal. Chem. 84, 304–311 (2012)CrossRefGoogle Scholar
  52. 52.
    Niles, R., Witkowska, H.E., Allen, S., Hall, S.C., Fisher, S.J., Hardt, M.: Acid-catalyzed oxygen-18 labeling of peptides. Anal. Chem. 81, 2804–2809 (2009)CrossRefGoogle Scholar
  53. 53.
    Liu, N., Wu, H., Liu, H., Chen, G., Cai, Z.: Microwave-assisted 18O-labeling of proteins catalyzed by formic acid. Anal. Chem. 82, 9122–9126 (2010)CrossRefGoogle Scholar
  54. 54.
    Parker, C.H., Stutts, W.L., DeGrasse, S.L.: Development and validation of a liquid chromatography-tandem mass spectrometry method for the quantitation of microcystins in blue-green algal dietary supplements. J. Agric. Food Chem. 63, 10303–10312 (2015)CrossRefGoogle Scholar
  55. 55.
    Robinson, N.A., Pace, J.G., Matson, C.F., Miura, G.A., Lawrence, W.B.: Tissue distribution, excretion and hepatic biotransformation of microcystin-LR in mice. J. Pharmacol. Exp. Therapeutics. 256, 176–182 (1991)Google Scholar
  56. 56.
    Buratti, F.M., Testai, E.: Species- and congener-differences in microcystin-LR and -RR GSH conjugation in human, rat, and mouse hepatic cytosol. Toxicol. Lett. 232, 133–140 (2015)CrossRefGoogle Scholar

Copyright information

© American Society for Mass Spectrometry 2018

Authors and Affiliations

  • Troy J. Attard
    • 1
    • 2
  • Melissa D. Carter
    • 3
  • Mengxuan Fang
    • 1
  • Rudolph C. Johnson
    • 3
  • Gavin E. Reid
    • 1
    • 2
    • 4
  1. 1.School of ChemistryThe University of MelbourneMelbourneAustralia
  2. 2.Bio21 Molecular Science and Biotechnology InstituteThe University of MelbourneMelbourneAustralia
  3. 3.Division of Laboratory SciencesNational Center for Environmental Health, Centers for Disease Control and PreventionAtlantaUSA
  4. 4.Department of Biochemistry and Molecular BiologyThe University of MelbourneMelbourneAustralia

Personalised recommendations