[en] Cyanobacteria are photosynthetic bacteria encountered in various aquatic environments. Some of them are able to produce powerful toxins called cyanotoxins. Among cyanotoxins, microcystins (MCs) constitute a group of closely related cyclic heptapeptides. Their sequences are made up of classical amino acids as well as post- translational modified ones. Interestingly, in vivo metabolism of microcystins seems to be greatly dependent on various minor structural differences and particularly those of the seventh amino acid, which can be either dehydroalanine (or a derivative), dehydroaminobutyric acid (or a derivative), serine or alanine. As a consequence, microcystins have been classified on the basis of the nature of this singular amino acid. A major difficulty in the classification of such toxins is that some of them share the same molecular masses and the same molecular formulas. Consequently, a simple mass measurement is not sufficient to determine the structure and the class of a toxin of interest. Heavy and expensive techniques are used to classify them, such as multi-dimensional nuclear magnetic resonance and amino acid analysis. In this work, a new matrix-assisted laser desorption/ionization time-of-flight method leading to an easy classification of MCs is proposed. The methodology relies on the reductive properties of the matrix 1,5-diaminonaphtalene (1,5-DAN) which appears to be able to selectively reduce the double carbon-carbon bond belonging to the seventh amino acid. Moreover, the yield of reduction seems to be influenced by the degree of substitution of this double bond, allowing a discrimination between dehydroalanine and dehydroaminobutyric acid. This selective reduction was confirmed by the study of three synthetic peptides by mass spectrometry and tandem mass spectrometry. According to these results, the use of reductive matrices seems to be promising in the study of microcystins and in their classification. More generally, 1,5-DAN allows the selective reduction of double carbon-carbon bonds. This property could also be employed in the characterization of others types of compound displaying double bonds (petrochemistry, metabolomics....).
Research Center/Unit :
Giga-Systems Biology and Chemical Biology - ULiège
I. Chorus and J. Bartram, in Toxic Cyanobacteria in Water: A guide to their Public Health Consequences, Monitoring and Management. E & F Spon, London, UK (1999).
WHO, Guidelines for Drinking-water Quality, Third Edition, Volume 1. Recommendations. World Health Organization, Geneva, Switzerland (2004).
J. Meriluoto and G.A. Codd, in Toxic Cyanobacterial Monitoring and Cyanotoxin Analysis. Äbo Akademi University Press, Aboensis, Finland (2005).
J. Fastner, I. Flieger, and U. Neumann, "Optimised extraction of microcystins from field samples-A comparison of different solvents and procedures", Water Res. 32, 3177-3181 (1998). doi: 10.1016/S0043-1354(98) 00073-6
M. Yuan, M. Namikoshi, A. Otsuki, K.L. Rinehart, K. Sivonen and M.F. Watanabe, "Low-energy collisionally activated decomposition and structural characterization of cyclic heptapeptide microcystins by electrospray ionization mass spectrometry", J. Mass Spectrom. 34, 33-43 (1999). doi: 10.1002/(SICI)1096-9888(199901)34:1<33::AID-JMS754>3.0.CO;2-L
R.M. Dawson, "The toxicology of microcystins", Toxicon 36, 953-962 (1998). doi: 10.1016/S0041-0101(97)00102-5
W.W. Carmichael, S.M.F.O. Azevedo, J.S. An, R.J.R. Molica, E.M. Jochimsen, S. Lau, K.L. Rinehart, G.R. Shaw and G.K. Eaglesham, "Human fatalities from cyanobacteria: Chemical and biological evidence for cyanotoxins", Environ. Health Persp. 109, 663-668 (2001). doi: 10.2307/3454781
K.A. Beattie, J. Ressler, C. Wiegand, E. Krause, G.A. Codd, C.E.W. Steinberg and S. Pflugmacher, "Comparative effects and metabolism of two microcystins and nodularin in the brine shrimp Artemia salina", Aquat. Toxicol. 62, 219-226 (2003). doi:10.1016/S0166-445X(02)00091-7
J.F. Blom and F. Juttner, "High crustacean toxicity of microcystin congeners does not correlate with high protein phosphatase inhibitory activity", Toxicon 46, 465-470 (2005). doi:10.1016/j.toxicon.2005.06.013
J.F. Blom, J.A. Robinson and F. Juttner, "High grazer toxicity of [D-Asp(3) (E)-Dhb(7)]microcystin-RR of Planktothrix rubescens as compared to different microcystins", Toxicon39, 1923-1932 (2001). doi: 10.1016/S0041-0101(01)00178-7
K. Kaya, T. Sano, H. Inoue and H. Takagi, "Selective determination of total normal microcystin by colorimetry, LC/UV detection and/or LC/MS", Anal. Chim. Acta 450, 73-80 (2001). doi: 10.1016/S0003-2670(01)01391-5
H. Ishii, M. Nishijima and T. Abe, "Characterization of degradation process of cyanobacterial hepatotoxins by a gram-negative aerobic bacterium", Water Res. 38, 2667-2676 (2004). doi: 10.1016/j.watres.2004.03. 014
S.J. Hoeger, D. Schmid. J.F. Blom, B. Ernst and D.R. Dietrich, "Analytical and functional characterization of microcystins [Asp(3)]MC-RR and [Asp[3),Dhb[7)]MC-RR: Consequences for risk assessment?", Environ. Sci. Technol. 41, 2609-2616 (2007). doi: 10.1021/es062681p
L.N. Sangolkar, S.S. Maske and T. Chakrabarti, "Methods for determining microcystins (peptide hepatotoxins) and microcystin-producing cyanobacteria", Water Res. 40, 3485-3496 (2006). doi: 10.1016/j.watres. 2006.08.010
R. Willame, T. Jurczak, J.F. Iffly, T. Kuli, J. Meriluoto and L. Hoffmann, "Distribution of hepatotoxic cyanobacterial blooms in Belgium and Luxembourg", Hydrobiologia 551, 99-117 (2005). doi: 10.1007/s10750-005- 4453-2
T. Sano, H. Takagi and K. Kaya, "A Dhb-microcystin from the filamentous cyanobacterium Planktothrix rubescens", Phytochemistry 65, 2159-2162 (2004). doi: 10.1016/j.phytochem.2004.03.034
H. Mazur-Marzec, L. Spoof, J. Kobos, M. Plinski and J. Meriluoto. "Cyanobacterial hepatotoxins, microcystins and nodularins, in fresh and brackish waters of the Pomeranian Province, northern Poland", Int. J. Oceanogr. Hydrobiol. 37(XXXVII), 3-21 (2008).
C. Hummert, J. Dahlmann, M. Reichelt and B. Luckas, "Analytical techniques for monitoring harmful cyanobacteria in lakes", Lakes & Reservoirs: Research and Management 6, 159-168 (2001). doi: 10.1046/J.1440-1770. 2001.00139.x
F. Hillenkamp, M. Karas, R.C. Beavis and B.T. Chait, "Matrix- assisted laser desorption ionization massspectrometry of biopolymers", Anal. Chem. 63, A1193-A1202 (1991). doi: 10.1021/ac00024a002
J.B. Fenn, M. Mann, C.K. Meng, S.F. Wong and C.M. Whitehouse, "Electrospray ionization for massspectrometry of large biomolecules", Science 246, 64-71 (1989). doi: 10.1126/science.2675315
G. Siuzdak, "The emergence of mass-spectrometry in biochemical-research", Proc. Nat. Acad. Sci. USA 91, 11290-11297 (1994). doi: 10.1073/pnas.91.24.11290
K. Demeure, L. Quinton. V. Gabelica and E. De Pauw, "Rational selection of the optimum MALDI matrix for top-down proteomics by in-source decay", Anal. Chem. 79, 8678-8685 (2007). doi: 10.1021/ac070849z
L. Quinton, K. Demeure, R. Dobson, N. Gilles, V. Gabelica and E. De Pauw, "New method for characterizing highly disulfide-bridged peptides in complex mixtures: Application to toxin identification from crude venoms", J. Proteome Res. 6, 3216-3223 (2007). doi: 10.1021/pr070142t
C. Ihling, K. Berger, M.M. Hofliger, D. Fuhrer. A.G. Beck-Sickingerand A. Sinz, "Nano-high-performance liquid chromatography in combination with nano-electrospray ionization Fourier transform ion-cyclotron resonance mass spectrometry for proteome analysis", Rapid Commun. Mass Spectrom. 17, 1240-1246 (2003). doi: 10.1002/rcm.1049
J. Wei, J.M. Buriak and G. Siuzdak, "Desorption-ionization mass spectrometry on porous silicon", Nature 399, 243-246 (1999). doi: 10.1038/20400
T. LeRiche, J. Osterodt and D.A. Volmer, "An experimental comparison of electrospray ion-trap and matrix-assisted laser desorption/ionization post-source decay mass spectra for the characterization of small drug molecules", Rapid Commun. Mass Spectrom. 15, 608-614 (2001). doi: 10.1002/rcm.278
K.L. Howard and G.L. Boyer, "Quantitative analysis of cyanobacterial toxins by matrix-assisted laser Desorption ionization mass Spectrometry", Anal. Chem. 79, 5980-5986 (2007). doi: 10.1021/ac0705723
L.H. Cohen and A.I. Gusev, "Small molecule analysis by MALDI mass spectrometry", Anal. Bioanal. Chem. 373, 571-586 (2002). doi: 10.1007/s00216-002-1321-z
M. Karas, M. Gluckmann and J. Schafer, "Ionization in matrix-assisted laser desorption/ionization: singly charged molecular ions are the lucky survivors", J. Mass Spectrom. 35, 1-12 (2000). doi: 10.1002/(SICI)1096-9888(200001)35:1<1::AID-JMS904>3.0.CO;2-0
M. Takayama, "In-source decay characteristics of peptides in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry", J. Am. Soc. Mass Spectrom. 12, 420-427 (2001). doi: 10.1016/S1044-0305(01)00218-5
M. Mann, P. Hojrup and P. Roepstorff, "Use of massspectrometric molecular-weight information to identify proteins in sequence databases", Biol. Mass Spectrom. 22, 338-345 (1993). doi: 10.1002/bms.1200220605
R.S. Johnson. S.A. Martin and K. Biemann, "Collision-induced fragmentation of (M + H)+ ions of peptides-side-chain specific sequence ions", Int. J. Mass Spectrom. Ion Proc. 86, 137-154 (1988). doi: 10.1016/0168-1176(88)80060-0
R.S. Johnson, S.A. Martin, K. Biemann, J.T. Stults and J.T. Watson, "Novel fragmentation process of peptides by collision-induced decomposition in a tandem mass-spectrometer-differentiation of leucine and isoleucine", Anal. Chem. 59, 2621-2625 (1987). doi: 10.1021/ac00148a019
V.H. Wysocki, K.A. Resing, Q.F. Zhang and G.L. Cheng, "Mass spectrometry of peptides and proteins", Methods 35, 211-222 (2005). doi: 10.1016/j.ymeth.2004.08.013
T. Sano and K. Kaya, "A 2-amino-2-butenoic acid(Dhb)-containing microcystin isolated from Oscillatoriaagardhii", Tetrahedron Letters 36, 8603-8606 (1995). doi:10.1016/0040-4039(95)01824-2
C.J. Hastie, E.B. Borthwick, L.F. Morrison, G.A. Codd and P.T.W. Cohen, "Inhibition of several protein phosphatases by a non-covalently interacting microcystin and a novel cyanobacterial peptide, nostocyclin", Biochim. Biophys. Acta-General Subjects 1726, 187-193 (2005). doi: 10.1016/j.bbagen.2005. 06.005
J.R. Bagu, B.D. Sykes, M.M. Craig and C.F.B. Holmes, "A molecular basis for different interactions of marine toxins with protein phosphatase-1-Molecular models for bound motuporin, microcystins, okadaic acid and calyculin A", J. Biol. Chem. 272, 5087-5097 (1997). doi:10.1074/jbc.272.8.5087
