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Dramatic improvement of proteomic analysis of zebrafish liver tumor by effective protein extraction with sodium deoxycholate and heat denaturation.

Wang J, Lee YM, Li C, Li P, Li Z, Lim TK, Gong Z, Lin Q - Int J Anal Chem (2015)

Bottom Line: The laborious gel-based approach is slowly being replaced by the advancing in-solution digestion approach.However, there are still several difficulties such as difficult-to-solubilize proteins, poor proteomic analysis in complex tissue samples, and the presence of sample impurities.Henceforth, there is a great demand to formulate a highly efficient protein extraction buffer with high protein extraction efficiency from tissue samples, high compatibility with in-solution digestion, reduced number of sample handling steps to reduce sample loss, low time consumption, low cost, and ease of usage.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, National University of Singapore, 14 Science Drive 4, Singapore 117543.

ABSTRACT
Majority of the proteomic studies on tissue samples involve the use of gel-based approach for profiling and digestion. The laborious gel-based approach is slowly being replaced by the advancing in-solution digestion approach. However, there are still several difficulties such as difficult-to-solubilize proteins, poor proteomic analysis in complex tissue samples, and the presence of sample impurities. Henceforth, there is a great demand to formulate a highly efficient protein extraction buffer with high protein extraction efficiency from tissue samples, high compatibility with in-solution digestion, reduced number of sample handling steps to reduce sample loss, low time consumption, low cost, and ease of usage. Here, we evaluated various existing protein extraction buffers with zebrafish liver tumor samples and found that sodium deoxycholate- (DOC-) based extraction buffer with heat denaturation was the most effective approach for highly efficient extraction of proteins from complex tissues such as the zebrafish liver tumor. A total of 4,790 proteins have been identified using shotgun proteomics approach with 2D LC, which to our knowledge is the most comprehensive study for zebrafish liver tumor proteome.

No MeSH data available.


Related in: MedlinePlus

A comparison between the identified proteins from SDS-heat- and DOC-heat-extracted samples. A total of 1,024 unique proteins were identified from 1D shotgun analysis.
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fig2: A comparison between the identified proteins from SDS-heat- and DOC-heat-extracted samples. A total of 1,024 unique proteins were identified from 1D shotgun analysis.

Mentions: Following the above observations, both SDS-heat-extracted (SDSΔX) and DOC-heat-extracted (DOCΔX) samples were subjected to 1D LC-MS/MS shotgun proteomics (1D shotgun) analysis to determine the number of proteins that could be identified. In our 1D shotgun results, 659 and 881 unique proteins were identified from SDSΔX and DOCΔX samples, respectively (Supplementary Tables  1 and 2) (see Supplementary Material available online at http://dx.doi.org/10.1155/2015/763969). Between the two sets of identified proteins, there were 516 overlapping proteins, with 143 and 365 proteins uniquely found in SDSΔX and DOCΔX samples, respectively (Figure 2). Therefore, the use of DOC-heat might be more effective than SDS-heat in extracting liver proteins or DOC-heat might have improved the downstream sample processing mainly including trypsin digestion and MS analysis.


Dramatic improvement of proteomic analysis of zebrafish liver tumor by effective protein extraction with sodium deoxycholate and heat denaturation.

Wang J, Lee YM, Li C, Li P, Li Z, Lim TK, Gong Z, Lin Q - Int J Anal Chem (2015)

A comparison between the identified proteins from SDS-heat- and DOC-heat-extracted samples. A total of 1,024 unique proteins were identified from 1D shotgun analysis.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4383156&req=5

fig2: A comparison between the identified proteins from SDS-heat- and DOC-heat-extracted samples. A total of 1,024 unique proteins were identified from 1D shotgun analysis.
Mentions: Following the above observations, both SDS-heat-extracted (SDSΔX) and DOC-heat-extracted (DOCΔX) samples were subjected to 1D LC-MS/MS shotgun proteomics (1D shotgun) analysis to determine the number of proteins that could be identified. In our 1D shotgun results, 659 and 881 unique proteins were identified from SDSΔX and DOCΔX samples, respectively (Supplementary Tables  1 and 2) (see Supplementary Material available online at http://dx.doi.org/10.1155/2015/763969). Between the two sets of identified proteins, there were 516 overlapping proteins, with 143 and 365 proteins uniquely found in SDSΔX and DOCΔX samples, respectively (Figure 2). Therefore, the use of DOC-heat might be more effective than SDS-heat in extracting liver proteins or DOC-heat might have improved the downstream sample processing mainly including trypsin digestion and MS analysis.

Bottom Line: The laborious gel-based approach is slowly being replaced by the advancing in-solution digestion approach.However, there are still several difficulties such as difficult-to-solubilize proteins, poor proteomic analysis in complex tissue samples, and the presence of sample impurities.Henceforth, there is a great demand to formulate a highly efficient protein extraction buffer with high protein extraction efficiency from tissue samples, high compatibility with in-solution digestion, reduced number of sample handling steps to reduce sample loss, low time consumption, low cost, and ease of usage.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, National University of Singapore, 14 Science Drive 4, Singapore 117543.

ABSTRACT
Majority of the proteomic studies on tissue samples involve the use of gel-based approach for profiling and digestion. The laborious gel-based approach is slowly being replaced by the advancing in-solution digestion approach. However, there are still several difficulties such as difficult-to-solubilize proteins, poor proteomic analysis in complex tissue samples, and the presence of sample impurities. Henceforth, there is a great demand to formulate a highly efficient protein extraction buffer with high protein extraction efficiency from tissue samples, high compatibility with in-solution digestion, reduced number of sample handling steps to reduce sample loss, low time consumption, low cost, and ease of usage. Here, we evaluated various existing protein extraction buffers with zebrafish liver tumor samples and found that sodium deoxycholate- (DOC-) based extraction buffer with heat denaturation was the most effective approach for highly efficient extraction of proteins from complex tissues such as the zebrafish liver tumor. A total of 4,790 proteins have been identified using shotgun proteomics approach with 2D LC, which to our knowledge is the most comprehensive study for zebrafish liver tumor proteome.

No MeSH data available.


Related in: MedlinePlus