A protocol for protein extraction from recalcitrant tissues of grapevine (Vitis vinifera L.) for proteome analysis

The difficulty in obtaining high quality proteins from recalcitrant plant species like grape (Vitis vinifera L.) is mainly due to low concentration of proteins, high activity of proteases and high level of interfering compounds, such as, pigments, polyphenols, tannins, flavonoids etc. Utilization of 2-dimentional electrophoresis (2DE) technique in proteome analysis largely depends on more efficient and optimized preparation of samples, which is regarded as one of the most important step for obtaining reliable information on proteins. In the present study, four different previously published protein extraction protocols and commercial protein extraction kits were compared to identify the most suitable protocol for protein extraction from different grape tissues, such as, leaves, buds, clusters and berries. Of the five protocols compared, resolution of protein in 1st dimension and 2nd dimension gel was improved with modified trichloro acetic acid/acetone precipitation with phenol extraction method. This method also yielded higher protein concentration, greater spot resolution with minimal streaking on 2DE gels of grape leaf protein. The same protocol also yielded higher quality protein from other grape tissues, such as, buds, berries and roots. Thus modified tricholoro acetic acid/acetone precipitation with phenol extraction method will be suitable for proteome analysis of different grapevine tissues.