Abstract
Three methods for extraction and preparation of high-quality proteins from both toxic and non-toxic dinoflagellates for proteomics analysis, including Trizol method, Lysis method and Tris method, were compared with the subsequent protein separation profiles using 2-D differential gel electrophoresis (2-D DIGE), Coomassie Blue and silver staining. These methods showed suitability for proteins with different pIs and molecular weights. Tris method was better for low molecular weight and low pI protein isolation; whereas both Lysis and Trizol method were better for high-molecular weight and high pI protein purification. Trizol method showed good results with Alexandrium species and Gynodinium species, and the background in gel was much clearer than the other two methods. At the same time, only Lysis method caused breaking down of the target proteins. On the other hand, Trizol method obtained higher concentration of ribulose-1,5-bisphosphate carboxylase/oxygenase proteins by Western-blotting, while Tris method was the best for peridinin-chlorophyll-protein complexes protein and T1 protein preparation. DIGE was better than Coomassie Blue and silver staining, except for some limitations, such as the high cost of the dyes, relatively short shelf life and the requirements for extensive and special image capturing equipment. Some proteins related to PSTs synthesis in dinoflagellates are hydrophobic with high molecular weight or binding on membranes and Trizol method performed better than Tris method for these proteins. The Trizol method and 2-D DIGE were effective combination for proteomics investigations of dinoflagellates. This procedure allows reliable and high recovery efficiency of proteins from dinoflagellates for better understanding on their occurrence and toxin-production for physiological and biochemical information.
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References
Cembella AD (1998) Ecophysiology and metabolism of paralytic shellfish toxins in marine microalgae. In: Anderson DM, Cembella AD, Hallegraeff GM (eds) Physiological ecology of harmful blooms. Springer, Berlin Heidelberg, pp 381–403
Chan LL, Lo SCL, Hodgkiss IJ (2002) Proteomic study of a model causative agent of harmful red tide, Prorocentrum triestrinum, I: optimization of sample preparation methodologies for analyzing with two-dimensional electrophoresis. Proteomics 2:1169–1186
Chan LL, Hodgkiss IJ, Lu S, Lo SCL (2004) Use of two-dimensional electrophoresis proteome reference maps of dinoflagellates for species recognition of causative agents of harmful algal blooms. Proteomics 4:180–192
Chan LL, Hodgkiss IJ, Lam PKS, Wan JMF, Chou H-N, Lum JHK, Lo MGY, Mak ASC, Sit W-H, Lo SCL (2005) Use of two-dimensional gel electrophoresis to differentiate morphospecies of Alexandrium minutum, a PSP toxin-producing agent of Harmful Algal Blooms. Proteomics 5:1580–1593
Chan LL, Sit W-H, Lam PKS, Hsieh DPH, Hodgkiss IJ, Wan JMF, Ho AYT, Choi NMC, Wang D-Z, Dudgeon D (2006) Identification and characterization of a ‘biomarker of toxicity’ from the proteome of the paralytic shellfish toxin-producing dinoflagellate, Alexandrium tamarense. Proteomics 6:654–666
Huang L, Zou Y, Weng H-W, Li H-Y, Liu J-S, Yang W-D (2015) Proteomic profile in Perna viridis after exposed to Prorocentrum lima, a dinoflagellate producing DSP toxins. Environ Pollut 196:350–357
Jiang X-W, Li X, Lam PKS, Cheng SH, Schlenk D, de Mitcheson YS, Li Y, Gu J-D, Chan LL (2012) Proteomic analysis of hepatic tissue of ciguatoxin (CTX) contaminated coral reef fish Cephalopholis argus and moray eel Gymnothorax undulates. Harmful Algae 13:65–71
Keller MD, Selvin RC, Claus W, Guillard RRL (1987) Media for the culture of oceanic ultraphytoplankton. J Phycol 23:633–638
Kellmann R, Stüken A, Orr RJS, Svendsen HM, Jakobsen KS (2010) Biosynthesis and molecular genetics of polyketides in marine dinoflagellates. Mar Drugs 8:1011–1048
Lee WF, Lo SCL (2008) The use of Trizol reagent (phenol/guanidine isothiocyanate) for producing high quality two-dimensional gel electrophoretograms (2-DE) of dinoflagellates. J Microbiol Methods 73:26–32
Lilley KS, Razzaq A, Dupree P (2002) Two-dimensional gelelectrophoresis: recent advances in sample preparation, detection and quantitation. Curr Opin Chem Biol 6:46–50
Molly MP, Herbert BR, Walsh BJ, Tyler MI, Traini M, SanchezJC Hochstrasser DF (1998) Extraction of membrane proteins by differential solubilization for separation using two-dimensional gel electrophoresis. Electrophoresis 19:837–844
Moustafa A, Loram JE, Hackett JD, Anderson DM, Plumley FG (2009) Origin of saxitoxin biosynthetic genes in cyanobacteria. PLoS One 4:e5758
Pomati F, Kellmann R, Cavalieri R, Burns BP, Neilan BA (2006) Comparative gene expression of PSP-toxin producing and non-toxic Anabaena circinalis strains. Environ Int 32:743–748
Sako Y, Yoshida T, Uchida A (2001) Purification and characterization of a sulfotransferase specific to N-21 of saxitoxin and gonyautoxin 2 + 3 from the toxic dinoflagellate Gymnodinium catenatum (Dinophyceae). J Phycol 37:1044–1051
Taroncher-Oldenbugr G, Anderson DM (2000) Identification and characterization of three differentially expressed genes, encoding S-adenosylhomocysteine hydrolase, methionine aminopeptidase, and a histone-like protein, in the toxic dinoflagellate Alexandrium fundyense. Appl Environ Microbiol 66:2105–2112
Tonge R, Shaw J, Middleton B, Rowlinson R, Rayner S, Young J, Pognan F, Hawkins E, Currie I, Davison M (2001) Validation and development of fluorescence two-dimensional differential gel electrophoresis proteomics technology. Proteomics 1:377–396
Unlu M, Morgan ME, Minden JS (1997) Difference gel electrophoresis: a single gel method for detecting changes in protein extracts. Electophoresis 18:2071–2077
Van den Bergh G, Arckens L (2004) Fluorescent two-dimensional differencegel electrophoresis unveils the potential of gel-based proteomics. Curr Opin Biotechnol 15:38–43
Wang DZ, Lin L, Chan LL, Hong HS (2009) Comparative studies of four protein preparation methods for proteomic study of the dinoflagellate Alexandrium sp. using two-dimensional electrophoresis. Harmful Algae 8:685–691
Wang DZ, Gao Y, Lin L, Hong H-S (2013) Comparative proteomic analysis reveals proteins putatively involved in toxin biosynthesis in the marine Dinoflagellate Alexandrium catenella. Mar Drugs 11:213–232
Zhang H, Lin S (2003) Complex gene structure of the form II RuBisCo in the dinoflagellate prorocentrum minimum (Dinophyceae). J Phycol 39:1160–1171
Acknowledgments
This research was supported by Hong Kong RGC CERG grant HKU765507M and PhD studentships from The University of Hong Kong. We are grateful to the laboratory assistance and support from Miss Jessie Lai.
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Xi-Wen Jiang and Jing Wang have contributed equally to this paper.
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Jiang, XW., Wang, J., Chan, L.L. et al. Comparison of three protein extraction procedures from toxic and non-toxic dinoflagellates for proteomics analysis. Ecotoxicology 24, 1395–1406 (2015). https://doi.org/10.1007/s10646-015-1514-9
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DOI: https://doi.org/10.1007/s10646-015-1514-9