Effective protein extraction protocol for proteomics studies of J erusalem artichoke leaves

Protein extraction is a crucial step for proteomics studies. To establish an effective protein extraction protocol suitable for two‐dimensional electrophoresis (2 DE ) analysis in J erusalem artichoke ( H elianthus tuberosus L .), three different protein extraction methods—trichloroacetic acid/acetone, M g/ NP ‐40, and phenol/ammonium acetate—were evaluated using J erusalem artichoke leaves as source materials. Of the three methods, trichloroacetic acid/acetone yielded the best protein separation pattern and highest number of protein spots in 2 DE analysis. Proteins highly abundant in leaves, such as R ubisco, are typically problematic during leaf 2 DE analysis, however, and this disadvantage was evident using trichloroacetic acid/acetone. To reduce the influence of abundant proteins on the detection of low‐abundance proteins, we optimized the trichloroacetic acid/acetone method by incorporating a PEG fractionation approach. After optimization, 363 additional (36.2%) protein spots were detected on the 2 DE gel. Our results suggest that trichloroacetic acid/acetone method is a better protein extraction technique than M g/ NP ‐40 and phenol/ammonium acetate in J erusalem artichoke leaf 2 DE analysis, and that trichloroacetic acid/acetone method combined with PEG fractionation procedure is the most effective approach for leaf 2 DE analysis of Jerusalem artichoke.