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

TY - JOUR

T1 - Comparison of three protein extraction procedures from toxic and non-toxic dinoflagellates for proteomics analysis

AU - Jiang, Xi Wen

AU - Wang, Jing

AU - Chan, Leo Lai

AU - Lam, Paul Kwan Sing

AU - Gu, Ji Dong

N1 - Publisher Copyright: © 2015, Springer Science+Business Media New York.

PY - 2015/8/27

Y1 - 2015/8/27

N2 - 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.

AB - 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.

KW - Dinoflagellates

KW - Lysis

KW - Paralytic shellfish toxins

KW - Protein extraction

KW - Proteomics

KW - Tris

KW - Trizol

UR - http://www.scopus.com/inward/record.url?scp=84938897647&partnerID=8YFLogxK

U2 - 10.1007/s10646-015-1514-9

DO - 10.1007/s10646-015-1514-9

M3 - 文章

C2 - 26197730

AN - SCOPUS:84938897647

SN - 0963-9292

VL - 24

SP - 1395

EP - 1406

JO - Ecotoxicology

JF - Ecotoxicology

IS - 6

ER -