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

• Published in: PloS one 2015-04, Vol.10 (4), p.e0124723-e0124723
• Main Authors: Hao, Ruijie, Adoligbe, Camus, Jiang, Bijie, Zhao, Xianlin, Gui, Linsheng, Qu, Kaixing, Wu, Sen, Zan, Linsen
• Format: Article
• Language: English
• Subjects: Acetone; Acetone - chemistry; Analysis; Animal sciences; Animals; Beef; Cattle; Centrifugation; Chemical Precipitation; Contaminants; Dissolution; Drying; Electrophoresis; Electrophoresis, Gel, Two-Dimensional - methods; Food; Galling; Gel electrophoresis; Genomes; Lipids; Lipids - chemistry; Methods; Muscle, Skeletal - chemistry; Muscles; Nucleic acids; Nucleic Acids - chemistry; Pollutant removal; Precipitates; Precipitation; Precipitation (Meteorology); Proteins; Proteins - chemistry; Proteome - analysis; Proteomics; Protocol; Red Meat - analysis; Trichloroacetic acid; Trichloroacetic Acid - chemistry; Two dimensional analysis; Zoology
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0124723

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.