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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

The major pathways involved in the molecular mechanisms of cancer as adapted from Ingenuity Pathway Analysis (IPA) database. The highlighted proteins in yellow depict our identified proteins from our 2D-LC-MS/MS analysis. A total of 77 proteins from our identified dataset from our 2D-LC-MS/MS are associated with the molecular mechanisms of cancer.
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fig5: The major pathways involved in the molecular mechanisms of cancer as adapted from Ingenuity Pathway Analysis (IPA) database. The highlighted proteins in yellow depict our identified proteins from our 2D-LC-MS/MS analysis. A total of 77 proteins from our identified dataset from our 2D-LC-MS/MS are associated with the molecular mechanisms of cancer.

Mentions: Further analysis using IPA revealed high coverage of our identified proteins in numerous canonical pathways. A close look into the pathways involved in the molecular mechanism of cancer identified a total of 77 associated proteins from our dataset, and Figure 5 shows the coverage of these proteins in the various cancer pathways. The coverage is relatively extensive, with many proteins identified upstream of pathways such as EGFR-Ras-mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase/protein kinase B (PI3K/AKT). The carcinogenesis of liver cancer consists of a complex, multifactorial, stepwise development [25]. These include genetic mutations affecting signaling pathway such as Wnt-β-catenin, hedgehog, hepatocyte growth factor/mesenchymal-epithelial transition factor (HGF/c-Met), insulin-like growth factor (IGF), PI3K/AKT/mammalian target of rapamycin (mTOR), MAPK, p53, phosphoretinoblastoma (pRb), Janus kinase-signal transducers and activators of transcription (JAK-STAT), and transforming growth factor-β (TGFβ) pathways [25–27].


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)

The major pathways involved in the molecular mechanisms of cancer as adapted from Ingenuity Pathway Analysis (IPA) database. The highlighted proteins in yellow depict our identified proteins from our 2D-LC-MS/MS analysis. A total of 77 proteins from our identified dataset from our 2D-LC-MS/MS are associated with the molecular mechanisms of cancer.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig5: The major pathways involved in the molecular mechanisms of cancer as adapted from Ingenuity Pathway Analysis (IPA) database. The highlighted proteins in yellow depict our identified proteins from our 2D-LC-MS/MS analysis. A total of 77 proteins from our identified dataset from our 2D-LC-MS/MS are associated with the molecular mechanisms of cancer.
Mentions: Further analysis using IPA revealed high coverage of our identified proteins in numerous canonical pathways. A close look into the pathways involved in the molecular mechanism of cancer identified a total of 77 associated proteins from our dataset, and Figure 5 shows the coverage of these proteins in the various cancer pathways. The coverage is relatively extensive, with many proteins identified upstream of pathways such as EGFR-Ras-mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase/protein kinase B (PI3K/AKT). The carcinogenesis of liver cancer consists of a complex, multifactorial, stepwise development [25]. These include genetic mutations affecting signaling pathway such as Wnt-β-catenin, hedgehog, hepatocyte growth factor/mesenchymal-epithelial transition factor (HGF/c-Met), insulin-like growth factor (IGF), PI3K/AKT/mammalian target of rapamycin (mTOR), MAPK, p53, phosphoretinoblastoma (pRb), Janus kinase-signal transducers and activators of transcription (JAK-STAT), and transforming growth factor-β (TGFβ) pathways [25–27].

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