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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

CBB stained gel from the 1D SDS-PAGE of proteins extracted from liver tumor samples using various extraction buffers. The loading concentration of each sample reflects the amount of proteins extracted from the liver samples before trypsin digestion. Larger number of protein bands would mean larger number of proteins extracted. Darker protein bands from each lane would mean a higher amount of proteins extracted. Black boxes indicate the two best extraction buffers and conditions in terms of number of protein bands and the intensity of the CBB stain. The highlighted regions for Lanes 2 and 7 show a larger number of visible bands compared to other lanes. Δ: heat.
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fig1: CBB stained gel from the 1D SDS-PAGE of proteins extracted from liver tumor samples using various extraction buffers. The loading concentration of each sample reflects the amount of proteins extracted from the liver samples before trypsin digestion. Larger number of protein bands would mean larger number of proteins extracted. Darker protein bands from each lane would mean a higher amount of proteins extracted. Black boxes indicate the two best extraction buffers and conditions in terms of number of protein bands and the intensity of the CBB stain. The highlighted regions for Lanes 2 and 7 show a larger number of visible bands compared to other lanes. Δ: heat.

Mentions: Following protein extraction from the harvested liver tumor tissue using the various extraction buffers, 1D SDS-PAGE analysis was performed to provide an initial visual indication of protein extraction efficiency. As shown in Figure 1, protein extraction using the DOC extraction buffer was potentially better than the other extraction buffers, as evident from the larger number of protein bands as well as higher intensity bands in the DOC-extracted protein lysate samples. Our results were comparable to a previous study conducted by Proc et al. [23], who demonstrated that both SDS and DOC were more superior denaturants than urea in terms of the greater amount of solubilized human plasma proteins. This could explain the larger number of protein bands in the liver tumor samples extracted with SDS or DOC. However, in extraction buffers like RIPA and TUTS, the concentration of SDS and DOC could be too low to obtain comparable results with those of SDS or DOC extraction buffers.


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)

CBB stained gel from the 1D SDS-PAGE of proteins extracted from liver tumor samples using various extraction buffers. The loading concentration of each sample reflects the amount of proteins extracted from the liver samples before trypsin digestion. Larger number of protein bands would mean larger number of proteins extracted. Darker protein bands from each lane would mean a higher amount of proteins extracted. Black boxes indicate the two best extraction buffers and conditions in terms of number of protein bands and the intensity of the CBB stain. The highlighted regions for Lanes 2 and 7 show a larger number of visible bands compared to other lanes. Δ: heat.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig1: CBB stained gel from the 1D SDS-PAGE of proteins extracted from liver tumor samples using various extraction buffers. The loading concentration of each sample reflects the amount of proteins extracted from the liver samples before trypsin digestion. Larger number of protein bands would mean larger number of proteins extracted. Darker protein bands from each lane would mean a higher amount of proteins extracted. Black boxes indicate the two best extraction buffers and conditions in terms of number of protein bands and the intensity of the CBB stain. The highlighted regions for Lanes 2 and 7 show a larger number of visible bands compared to other lanes. Δ: heat.
Mentions: Following protein extraction from the harvested liver tumor tissue using the various extraction buffers, 1D SDS-PAGE analysis was performed to provide an initial visual indication of protein extraction efficiency. As shown in Figure 1, protein extraction using the DOC extraction buffer was potentially better than the other extraction buffers, as evident from the larger number of protein bands as well as higher intensity bands in the DOC-extracted protein lysate samples. Our results were comparable to a previous study conducted by Proc et al. [23], who demonstrated that both SDS and DOC were more superior denaturants than urea in terms of the greater amount of solubilized human plasma proteins. This could explain the larger number of protein bands in the liver tumor samples extracted with SDS or DOC. However, in extraction buffers like RIPA and TUTS, the concentration of SDS and DOC could be too low to obtain comparable results with those of SDS or DOC extraction buffers.

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