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An Optimized Trichloroacetic Acid/Acetone Precipitation Method for Two-Dimensional Gel Electrophoresis Analysis of Qinchuan Cattle Longissimus Dorsi Muscle Containing High Proportion of Marbling.

Hao R, Adoligbe C, Jiang B, Zhao X, Gui L, Qu K, Wu S, Zan L - PLoS ONE (2015)

Bottom Line: Unfortunately, the vast amounts of lipids and nucleic acids in this tissue hampered LD proteomics analysis.According to our result, the treatment for solid precipitate facilitated non-protein contaminant removal and protein re-dissolution, ultimately improving two-dimensional gel electrophoresis (2-DE) analysis.It was found that 30 min air-drying did not result in significant protein loss, but reduced horizontal streaking and smearing on 2-DE gel compared to 10 min.

View Article: PubMed Central - PubMed

Affiliation: College of Animal Science and Technology, Northwest A&F University, Yangling, Shaanxi, P.R. China, 712100.

ABSTRACT
Longissimus dorsi muscle (LD) proteomics provides a novel opportunity to reveal the molecular mechanism behind intramuscular fat deposition. Unfortunately, the vast amounts of lipids and nucleic acids in this tissue hampered LD proteomics analysis. Trichloroacetic acid (TCA)/acetone precipitation is a widely used method to remove contaminants from protein samples. However, the high speed centrifugation employed in this method produces hard precipitates, which restrict contaminant elimination and protein re-dissolution. To address the problem, the centrifugation precipitates were first grinded with a glass tissue grinder and then washed with 90% acetone (TCA/acetone-G-W) in the present study. According to our result, the treatment for solid precipitate facilitated non-protein contaminant removal and protein re-dissolution, ultimately improving two-dimensional gel electrophoresis (2-DE) analysis. Additionally, we also evaluated the effect of sample drying on 2-DE profile as well as protein yield. It was found that 30 min air-drying did not result in significant protein loss, but reduced horizontal streaking and smearing on 2-DE gel compared to 10 min. In summary, we developed an optimized TCA/acetone precipitation method for protein extraction of LD, in which the modifications improved the effectiveness of TCA/acetone method.

No MeSH data available.


Related in: MedlinePlus

Overlay analysis of total protein spots with different methods.A: comparison of TCA/acetone-G-W to direct extraction; B: comparison of TCA/acetone-G-W to two standard TCA/acetone procedures. U represented the number of unique spots from each method; C represented the number of spots common to all the methods.
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pone.0124723.g002: Overlay analysis of total protein spots with different methods.A: comparison of TCA/acetone-G-W to direct extraction; B: comparison of TCA/acetone-G-W to two standard TCA/acetone procedures. U represented the number of unique spots from each method; C represented the number of spots common to all the methods.

Mentions: To further understand our modified protocol, overlay analysis of TCA/acetone-G-W to direct extraction and to two standard TCA/acetone methods regarding spot pattern were conducted respectively. As shown in Fig 2A, there were 533 protein spots present in both direct extraction and TCA/acetone-G-W. The number of unique spots in direct extraction was 81, far less than TCA/acetone-G-W(535). The result wasn’t surprising, since many spots were covered owning to low resolution, high background, serious streaking and smearing in gels. When we analyzed spot pattern of direct extraction with PDQuest software, it was difficult to distinguish a real spot from streaking and smearing due to blurry edges, going against the following steps such as spot incision and identification with MS. Thus contaminant elimination was much necessary for 2-DE gel with high resolution and clean background in LD proteomics. Fig 2B delivered the comparison of protein spots between TCA/acetone methods. 383 protein spots existed in all the three procedures; 511 spots were common to spot patterns of TCA/acetone and TCA/acetone-G-W, which accounted for 91.4% of all spots with TCA/acetone. Parallelly, 568 spots simultaneously occurred in TCA/acetone-B and TCA/acetone-G-W, taking up 90.0% of all spots with TCA/acetone-B. The total number of unique proteins detected by TCA/acetone-G-W was the highest (371), far exceeding the corresponding number for TCA/acetone-B (30) and TCA/acetone (15). The above results implied that TCA/acetone-G-W not only captured the most of proteins present in common TCA/acetone extracts, but also extracted many novel proteins. The increasing number of detected protein spots should be attributed to better spot focusing and more extracted proteins in TCA/acetone-G-W.


An Optimized Trichloroacetic Acid/Acetone Precipitation Method for Two-Dimensional Gel Electrophoresis Analysis of Qinchuan Cattle Longissimus Dorsi Muscle Containing High Proportion of Marbling.

Hao R, Adoligbe C, Jiang B, Zhao X, Gui L, Qu K, Wu S, Zan L - PLoS ONE (2015)

Overlay analysis of total protein spots with different methods.A: comparison of TCA/acetone-G-W to direct extraction; B: comparison of TCA/acetone-G-W to two standard TCA/acetone procedures. U represented the number of unique spots from each method; C represented the number of spots common to all the methods.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4404140&req=5

pone.0124723.g002: Overlay analysis of total protein spots with different methods.A: comparison of TCA/acetone-G-W to direct extraction; B: comparison of TCA/acetone-G-W to two standard TCA/acetone procedures. U represented the number of unique spots from each method; C represented the number of spots common to all the methods.
Mentions: To further understand our modified protocol, overlay analysis of TCA/acetone-G-W to direct extraction and to two standard TCA/acetone methods regarding spot pattern were conducted respectively. As shown in Fig 2A, there were 533 protein spots present in both direct extraction and TCA/acetone-G-W. The number of unique spots in direct extraction was 81, far less than TCA/acetone-G-W(535). The result wasn’t surprising, since many spots were covered owning to low resolution, high background, serious streaking and smearing in gels. When we analyzed spot pattern of direct extraction with PDQuest software, it was difficult to distinguish a real spot from streaking and smearing due to blurry edges, going against the following steps such as spot incision and identification with MS. Thus contaminant elimination was much necessary for 2-DE gel with high resolution and clean background in LD proteomics. Fig 2B delivered the comparison of protein spots between TCA/acetone methods. 383 protein spots existed in all the three procedures; 511 spots were common to spot patterns of TCA/acetone and TCA/acetone-G-W, which accounted for 91.4% of all spots with TCA/acetone. Parallelly, 568 spots simultaneously occurred in TCA/acetone-B and TCA/acetone-G-W, taking up 90.0% of all spots with TCA/acetone-B. The total number of unique proteins detected by TCA/acetone-G-W was the highest (371), far exceeding the corresponding number for TCA/acetone-B (30) and TCA/acetone (15). The above results implied that TCA/acetone-G-W not only captured the most of proteins present in common TCA/acetone extracts, but also extracted many novel proteins. The increasing number of detected protein spots should be attributed to better spot focusing and more extracted proteins in TCA/acetone-G-W.

Bottom Line: Unfortunately, the vast amounts of lipids and nucleic acids in this tissue hampered LD proteomics analysis.According to our result, the treatment for solid precipitate facilitated non-protein contaminant removal and protein re-dissolution, ultimately improving two-dimensional gel electrophoresis (2-DE) analysis.It was found that 30 min air-drying did not result in significant protein loss, but reduced horizontal streaking and smearing on 2-DE gel compared to 10 min.

View Article: PubMed Central - PubMed

Affiliation: College of Animal Science and Technology, Northwest A&F University, Yangling, Shaanxi, P.R. China, 712100.

ABSTRACT
Longissimus dorsi muscle (LD) proteomics provides a novel opportunity to reveal the molecular mechanism behind intramuscular fat deposition. Unfortunately, the vast amounts of lipids and nucleic acids in this tissue hampered LD proteomics analysis. Trichloroacetic acid (TCA)/acetone precipitation is a widely used method to remove contaminants from protein samples. However, the high speed centrifugation employed in this method produces hard precipitates, which restrict contaminant elimination and protein re-dissolution. To address the problem, the centrifugation precipitates were first grinded with a glass tissue grinder and then washed with 90% acetone (TCA/acetone-G-W) in the present study. According to our result, the treatment for solid precipitate facilitated non-protein contaminant removal and protein re-dissolution, ultimately improving two-dimensional gel electrophoresis (2-DE) analysis. Additionally, we also evaluated the effect of sample drying on 2-DE profile as well as protein yield. It was found that 30 min air-drying did not result in significant protein loss, but reduced horizontal streaking and smearing on 2-DE gel compared to 10 min. In summary, we developed an optimized TCA/acetone precipitation method for protein extraction of LD, in which the modifications improved the effectiveness of TCA/acetone method.

No MeSH data available.


Related in: MedlinePlus