Optimization of protein extraction and solubilization for mature grape berry clusters

Abstract

Protein extraction from grape berries has been challenging, particularly in mature berries, which can have sugar concentrations as high as 26%. Grape skins and seeds contain large amounts of polyphenols, which can also interfere with efficient protein extraction. In plants, two extraction protocols, TCA/acetone-based and phenol-based methods, have been mainly used to extract proteins from different organs or tissues on many species. However, few results have been reported for grape berry clusters. We wanted to determine which of these protocols was optimal for berry clusters in order to achieve both efficient protein extraction and high spot resolution on 2-D gels. Four protocols, derived from either TCA/acetone or phenol procedures, were tested on mature Cabernet Sauvignon whole berry clusters. The phenol-based protocols were superior to the TCA/acetone methods, showing larger protein yields and greater spot resolution on 2-D gels. One method was clearly superior to the rest, a phenol-based extraction method combined with resuspension in the presence of both urea and thiourea as chaotropes. A total of 81 spots were excised and identified following MALDI-TOF/TOF MS analyses. Their identification helped further characterize the specificity of each extraction procedure.

Protein extraction from Phoenix dactylifera L. leaves, a recalcitrant material, for two‐dimensional electrophoresis

“…They also reduce polysaccharide content [4]. Although comparative studies are scarce, Ph-based protocols seem to be superior to TCA/acetone methods for protein extraction from recalcitrant plant tissues [4,21]. Nevertheless, both methods should be tested for a given recalcitrant tissue [5].…”

A protein extraction method compatible with proteomic analysis for the euhalophyte Salicornia europaea

“…Using this method, proteins are solubilized in Phe and subsequently precipitated with methanol and ammonium acetate [3,13,[15][16][17]. Recently, Phe-based methods were modified specifically for 2-DE when dealing with various recalcitrant plant tissues, such as olive leaf [17], cotton fibers [18], mature grape berry clusters [19], banana meristem cultures, meristems of apple plantlets, and leaves of potato plantlets [15].…”

Identification of proteins from wild cardoon flowers (Cynara cardunculus L.) by a proteomic approach

“…Several studies on potato, banana, mature grape [25], apple, and strawberry fruits [26], using this method, resulted in higher protein yields and greater spot resolutions. In order to understand this higher efficiency [27] compared absorption spectra of protein extracts in IEF buffer, stemming from three different extractions, which were applied on Beta vulgaris tissues.…”

“…Furthermore, three spots were found at low molecular levels, with an approximate molecular mass in the range of 10 and 20 kDa. In fact, according to several studies of cardoon's protein profile [14][15][16][17][18][19][20][21][22][23][24][25][26][27][28][29][30][31], proteins with low Mw, represented the β-subunits of cardosins (16.5 and 13.5 kDa), since there were a positive correlation between band intensities and milk-clotting activities.…”