Abstract
Thaumatin-like proteins (TLPs) and chitinases are the main constituents of so-called protein hazes which can form in finished white wine and which is a great concern of winemakers. These soluble pathogenesis-related (PR) proteins are extracted from grape berries. However, their distribution in different grape tissues is not well documented. In this study, proteins were first separately extracted from the skin, pulp and seed of Sauvignon Blanc grapes, followed by trypsin digestion and analysis by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS). Proteins identified included 75 proteins from Sauvignon Blanc grape skin, 63 from grape pulp and 35 from grape seed, mostly functionally classified as associated with metabolism and energy. Some were present exclusively in specific grape tissues; for example, proteins involved in photosynthesis were only detected in grape skin and proteins found in alcoholic fermentation were only detected in grape pulp. Moreover, proteins identified in grape seed were less diverse than those identified in grape skin and pulp. TLPs and chitinases were identified in both Sauvignon Blanc grape skin and pulp, but not in the seed. To relatively quantify the PR proteins, the protein extracts of grape tissues were seperated by HPLC first and then analysed by SDS-PAGE. The results showed that the protein fractions eluted at 9.3 min and 19.2 min under the chromatographic conditions of this study confirmed that these corresponded to TLPs and chitinases seperately. Thus, the relative quantification of TLPs and chitinases in protein extracts was carried out by comparing the area of corresponding peaks against the area of a thamautin standard. The results presented in this study clearly demonstrated the distribution of haze-forming PR proteins in grape berries, and the relative quantification of TLPs and chitinases could be applied in fast tracking of changes in PR proteins during grape growth and determination of PR proteins in berries at harvest.
Highlights
Protein stabilization of white wine is of great concern to winemakers as denaturation of proteins in wine may cause haze formation, which is usually considered a wine fault
Pathogenesis-related (PR) proteins originally derived from grape berries are the major soluble proteins remaining in finished wine and they are mainly responsible for haze formation [1,2]
This direct injection method provides rapid identification of the major proteins extracted from grape tissues, but high intensity peaks from liquid chromatography separation may eclipse lower intensity peaks due to the lack of pre-fractionation, so proteins with low concentration in protein mixture may be less encountered and identified in whole tissue proteomic studies
Summary
Protein stabilization of white wine is of great concern to winemakers as denaturation of proteins in wine may cause haze formation, which is usually considered a wine fault. On the basis of similarities in amino acid sequences, serological relationship, and/or enzymatic or biological activity, eleven families have been recognized and classified for tobacco and tomato [5] Some of these PR protein family members have been found in grapevine. With the accomplishment of grapevine genome sequencing programmes in 2007 [11,12] and the development of technology in protein analysis, proteomic analysis of grapevine has significantly improved knowledge of grape proteins and produced a better understanding of their characteristics [13] These have identified other PR protein family members found in grapevine, such as osmotins (PR-5 family), β-1,3-glucanases (PR-2 family) and the PR-10 proteins [14,15,16]
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