Limits...
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 PI3K/AKT/mTOR pathway 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 79 proteins from our identified dataset from our 2D-LC-MS/MS are associated with this pathway.
© Copyright Policy - open-access
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4383156&req=5

fig6: The PI3K/AKT/mTOR pathway 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 79 proteins from our identified dataset from our 2D-LC-MS/MS are associated with this pathway.

Mentions: Two of the fundamental hallmarks of cancers are the ability to sustain prolonged cell proliferation and the use of abnormal metabolic pathways to generate energy. The PI3K/AKT/mTOR pathway is documented to regulate cell growth, aging, and metabolism [28]. Furthermore, our previous study also identified the presence of dysregulated PI3K/AKT/mTOR pathway in xmrk zebrafish liver tumors [29]. It has been documented that this pathway is upregulated in 40–50% of hepatocellular carcinoma, which is the most common primary cancer of the liver [30–32]. Henceforth, we subjected our 2D shotgun dataset to pathway analysis using IPA. Our results showed that 79 associated proteins were identified belonging to the PI3K/AKT/mTOR pathway, notably Ras, PI3K, AKT, mTOR, p70S6K, 4EBP, and eIF4E proteins (Figure 6). With such a high coverage of proteins associated with the PI3K/AKT/mTOR pathway, it would be highly beneficial for further studies pertaining to this pathway in relation to liver cancer. This again demonstrates the potential of our DOC-based protein extraction method in high-throughput proteomic studies to elucidate the molecular progression of diseases.


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 PI3K/AKT/mTOR pathway 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 79 proteins from our identified dataset from our 2D-LC-MS/MS are associated with this pathway.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig6: The PI3K/AKT/mTOR pathway 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 79 proteins from our identified dataset from our 2D-LC-MS/MS are associated with this pathway.
Mentions: Two of the fundamental hallmarks of cancers are the ability to sustain prolonged cell proliferation and the use of abnormal metabolic pathways to generate energy. The PI3K/AKT/mTOR pathway is documented to regulate cell growth, aging, and metabolism [28]. Furthermore, our previous study also identified the presence of dysregulated PI3K/AKT/mTOR pathway in xmrk zebrafish liver tumors [29]. It has been documented that this pathway is upregulated in 40–50% of hepatocellular carcinoma, which is the most common primary cancer of the liver [30–32]. Henceforth, we subjected our 2D shotgun dataset to pathway analysis using IPA. Our results showed that 79 associated proteins were identified belonging to the PI3K/AKT/mTOR pathway, notably Ras, PI3K, AKT, mTOR, p70S6K, 4EBP, and eIF4E proteins (Figure 6). With such a high coverage of proteins associated with the PI3K/AKT/mTOR pathway, it would be highly beneficial for further studies pertaining to this pathway in relation to liver cancer. This again demonstrates the potential of our DOC-based protein extraction method in high-throughput proteomic studies to elucidate the molecular progression of diseases.

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