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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

Subcellular localization of the identified proteins based on GO analysis. (a) 881 proteins identified in DOCΔX samples; (b) 659 proteins identified in SDSΔX samples generated using STRAP. The identified subcellular localization profiles are largely similar between both samples, with only the DOCΔX samples having proteins located in the endosome. The larger number of proteins identified from the DOCΔX samples also showed more even distribution of proteins across all subcellular locations. Percentages are rounded to the nearest whole number.
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fig3: Subcellular localization of the identified proteins based on GO analysis. (a) 881 proteins identified in DOCΔX samples; (b) 659 proteins identified in SDSΔX samples generated using STRAP. The identified subcellular localization profiles are largely similar between both samples, with only the DOCΔX samples having proteins located in the endosome. The larger number of proteins identified from the DOCΔX samples also showed more even distribution of proteins across all subcellular locations. Percentages are rounded to the nearest whole number.

Mentions: To determine whether different proteins identified from the two groups differ in terms of their subcellular localization, we conducted a GO analysis of the proteins using the D. rerio GOSLIM database. In the GO analysis of SDSΔX and DOCΔX samples, majority of the proteins are found in the cytoplasm (20% and 25% resp.; Figure 3). The subcellular localization profiles of both samples are observed to be very similar, but the additional proteins identified from the DOCΔX samples resulted in more even distribution of protein in the various subcellular locations, even detecting proteins in the endosome, which is absent from the SDSΔX sample. Our results highlighted the comparability of DOC- and SDS-based extraction method in their proteome coverage.


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)

Subcellular localization of the identified proteins based on GO analysis. (a) 881 proteins identified in DOCΔX samples; (b) 659 proteins identified in SDSΔX samples generated using STRAP. The identified subcellular localization profiles are largely similar between both samples, with only the DOCΔX samples having proteins located in the endosome. The larger number of proteins identified from the DOCΔX samples also showed more even distribution of proteins across all subcellular locations. Percentages are rounded to the nearest whole number.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig3: Subcellular localization of the identified proteins based on GO analysis. (a) 881 proteins identified in DOCΔX samples; (b) 659 proteins identified in SDSΔX samples generated using STRAP. The identified subcellular localization profiles are largely similar between both samples, with only the DOCΔX samples having proteins located in the endosome. The larger number of proteins identified from the DOCΔX samples also showed more even distribution of proteins across all subcellular locations. Percentages are rounded to the nearest whole number.
Mentions: To determine whether different proteins identified from the two groups differ in terms of their subcellular localization, we conducted a GO analysis of the proteins using the D. rerio GOSLIM database. In the GO analysis of SDSΔX and DOCΔX samples, majority of the proteins are found in the cytoplasm (20% and 25% resp.; Figure 3). The subcellular localization profiles of both samples are observed to be very similar, but the additional proteins identified from the DOCΔX samples resulted in more even distribution of protein in the various subcellular locations, even detecting proteins in the endosome, which is absent from the SDSΔX sample. Our results highlighted the comparability of DOC- and SDS-based extraction method in their proteome coverage.

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