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

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.

Fig 1. Representative 2-DE maps of LD protein extracted using different methods.

A: one-step extraction by lysis buffer; B: TCA/acetone; C: TCA/acetone-B; D: TCA/acetone-G-W. The modified procedure of TCA/acetone-G-W gave the best 2-DE gel with a clear background and minimal streaking and smearing.

Fig 2. 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.