Comparison of protein precipitation methods from adipose tissue using difference gel electrophoresis

Abstract

Proteomic methods have great potential to aid our understanding of the functional and pathological roles of adipose tissue. A critical initial step in the proteomic studies is the efficient isolation of proteins before conducting detailed analysis. In this study, three different methods were used for precipitating proteins; we analyzed samples from visceral adipose tissue, subcutaneous adipose tissue, and stromal visceral fraction extracts after chloroform/methanol, acetone, and trichloroacetic acid precipitation. The proteins recovered after the precipitation steps were examined by 2D-DIGE. Statistical analyses were carried out using simple linear regression analyses and R2 values were calculated for the intra- and inter-method comparisons. We found that all three precipitation methods provided highly reproducible protein spots that were recovered when run in duplicate using the same method of precipitation, irrespective of whether it was solvent (R2 = 0.85-0.98) or acid-based (R2 = 0.80-0.96). A higher variation and poor correlation was noted for the recovered protein spots when comparisons were made between the methods (R2 = 0.40-0.88) and also when the same method was compared between different sample types. In this study, TCA-precipitated samples were enriched in lower molecular mass proteins compared to the samples extracted by solvent-based precipitation methods.

Keywords: 2D-DIGE; Adipose tissue; Comparison of method; Protein precipitation; Proteomics.