Proteomic analysis of hepatic tissue of ciguatoxin (CTX) contaminated coral reef fish Cephalopholis argus and moray eel Gymnothorax undulatus

Jiang, Xi-Wen; Li, Xiaomin; Cheng, Shuk Han; Schlenk, Daniel; Mitcheson, Yvonne Sadovy de; Liu, Ying; Gu, Ji‐Dong; Chan, Leo Lai

doi:10.1016/j.hal.2011.10.009

Cited by 18 publications

(19 citation statements)

References 32 publications

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“…The procedures were the same as described by Jiang et al (2012). Equal amount (50 lg) of samples were labeled with Cy3 and Cy5.…”

Section: Dige Of Protein Samplesmentioning

confidence: 99%

“…The procedures were described previously (Jiang et al 2012). In silver staining and Coomassie Blue staining with 2-D electrophoresis of each sample protein extracts, the first dimension was carried out on IPG strips (GE healthcare Life Sciences, Psicataway, New Jersey) in the Ettan IPGphor III System (GE healthcare) with running conditions as follows: 50 V for 13 h; 50 to 100 V for 2 h; 100-200 V for 2 h; 200-500 V for 1 h; 500-1000 V for 2 h; 1000-4000 V for 2 h, 8000 V up to total volt-hours reached 50 kVh.…”

Section: Two-dimensional Electrophoresis (2-de) Analysis By Silver Anmentioning

confidence: 99%

“…The different protein spots from any of the two comparisons were identified by MALDI-TOF/TOF MS as described elsewhere (Jiang et al 2012). Proteins with over 1.5-fold change threshold were manually selected and washed with water (three times), buffer (once) containing 100 mM of Na 2 S 2 O 3 and 30 mM of K 3 [Fe(CN) 6 ], and then washed again with water (three times), each for 15 min.…”

Section: Identification Of Different Spots In Proteomics Analysismentioning

confidence: 99%

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Comparison of three protein extraction procedures from toxic and non-toxic dinoflagellates for proteomics analysis

et al. 2015

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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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“…The procedures were the same as described by Jiang et al (2012). Equal amount (50 lg) of samples were labeled with Cy3 and Cy5.…”

Section: Dige Of Protein Samplesmentioning

confidence: 99%

Section: Two-dimensional Electrophoresis (2-de) Analysis By Silver Anmentioning

confidence: 99%

Section: Identification Of Different Spots In Proteomics Analysismentioning

confidence: 99%

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Comparison of three protein extraction procedures from toxic and non-toxic dinoflagellates for proteomics analysis

et al. 2015

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“…Other steps of the CBA-N2a have also been the subject of substantial modifications. For instance, although many studies used a 500/50 µM (1:10 ratio) for O/V treatment [32][33][34][35][36][37][38][39][40][41][42][43][44][45][46][47][48][49]76,89,91,92] as initially defined by Manger et al [19,20,30,31], numerous changes in O/V concentrations [54][55][56][57]59,61,[63][64][65][71][72][73][74][75][77][78][79][80][81][82][83]85,87,[93]…”

Section: Introductionmentioning

confidence: 99%

Revisiting the Neuroblastoma Cell-Based Assay (CBA-N2a) for the Improved Detection of Marine Toxins Active on Voltage Gated Sodium Channels (VGSCs)

Viallon

Chinain

Darius

2020

Toxins

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The neuroblastoma cell-based assay (CBA-N2a) is widely used for the detection of marine biotoxins in seafood products, yet a consensus protocol is still lacking. In this study, six key parameters of CBA-N2a were revisited: cell seeding densities, cell layer viability after 26 h growth, MTT incubation time, Ouabain and Veratridine treatment and solvent and matrix effects. A step-by-step protocol was defined identifying five viability controls for the validation of CBA-N2a results. Specific detection of two voltage gated sodium channel activators, pacific ciguatoxin (P-CTX3C) and brevetoxin (PbTx3) and two inhibitors, saxitoxin (STX) and decarbamoylsaxitoxin (dc-STX) was achieved, with EC50 values of 1.7 ± 0.35 pg/mL, 5.8 ± 0.9 ng/mL, 3 ± 0.5 ng/mL and 15.8 ± 3 ng/mL, respectively. When applied to the detection of ciguatoxin (CTX)-like toxicity in fish samples, limit of detection (LOD) and limit of quantification (LOQ) values were 0.031 ± 0.008 and 0.064 ± 0.016 ng P-CTX3C eq/g of flesh, respectively. Intra and inter-assays comparisons of viability controls, LOD, LOQ and toxicity in fish samples gave coefficients of variation (CVs) ranging from 3% to 29%. This improved test adaptable to either high throughput screening or composite toxicity estimation is a useful starting point for a standardization of the CBA-N2a in the field of marine toxin detection.

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“…One was 12 h after a normal L/D cycle that produced low toxin samples (12 h, 20:00 pm)-1, and the other was 22 h after a normal L/D cycle representing high toxin sample (22 h, 06:00 am)-2. Proteins were extracted, labeled, separated, and finally identified by MALDI-TOF/TOF MS as described by Jiang et al (2012).…”

Section: Proteomics and Western Blot Analysis Of T1 Proteinsmentioning

confidence: 99%

Relationship of proteomic variation and toxin synthesis in the dinoflagellate Alexandrium tamarense CI01 under phosphorus and inorganic nitrogen limitation

et al. 2015

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Paralytic shellfish toxins (PSTs) are originated from cyanobacteria and dinoflagellates, including Alexandrium tamarense, the common dinoflagellate species. In this study, a toxic dinoflagellate strain of A. tamarense CI01 was selected for studying the PSTs' concentration and the related protein variation during the whole cell cycle under different nutrient conditions. High-performance liquid chromatography, 2-D DIGE and Western blotting were used collectively for protein profiling and identification. Results showed that the toxin content was suppressed under nitrogen limiting condition, but enhanced in phosphorous limiting medium. Based on the results of proteomics analysis, 7 proteins were discovered to be related to the PSTs biosynthesis of A. tamarense CI01, including S-adenosylhomocysteine hydrolase, ornithine cyclodeaminase, argininosuccinate synthase, methyluridine methyltransferase cystine ABC transporter, phosphoserine phosphatase, argininosuccinate synthase and acyl-CoA dehydrogenase, which corresponds to the metabolism of the methionine, cysteine, ornithine, arginine and proline. Moreover, some photosynthesis relating proteins also increased their expression during PST synthesis period in A. tamarense CI01, such as phosphoenolpyruvate carboxylase, chloroplast phosphoglycerate kinase, peridinin-chlorophyll α-binding protein, Mg(2+) transporter protein and chloroplast phosphoglycerate kinase. The above findings are in support of our hypothesis that these proteins are involved in toxin biosynthesis of A. tamarense CI01, but cause-and-effect mechanisms need to be investigated in further studies.

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Proteomic analysis of hepatic tissue of ciguatoxin (CTX) contaminated coral reef fish Cephalopholis argus and moray eel Gymnothorax undulatus

Cited by 18 publications

References 32 publications

Comparison of three protein extraction procedures from toxic and non-toxic dinoflagellates for proteomics analysis

Comparison of three protein extraction procedures from toxic and non-toxic dinoflagellates for proteomics analysis

Revisiting the Neuroblastoma Cell-Based Assay (CBA-N2a) for the Improved Detection of Marine Toxins Active on Voltage Gated Sodium Channels (VGSCs)

Relationship of proteomic variation and toxin synthesis in the dinoflagellate Alexandrium tamarense CI01 under phosphorus and inorganic nitrogen limitation

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