Proteomic Analysis of Sauvignon Blanc Grape Skin, Pulp and Seed and Relative Quantification of Pathogenesis-Related Proteins

RESEARCH ARTICLE Proteomic Analysis of Sauvignon Blanc Grape Skin, Pulp and Seed and Relative Quantification of Pathogenesis-Related Proteins Bin Tian1*, Roland Harrison1, James Morton1, Santanu Deb-Choudhury2 1 Department of Wine, Food and Molecular Biosciences, Lincoln University, Lincoln, 7647, Canterbury, New Zealand, 2 Agresearch, Lincoln Research Centre, Christchurch, 8140, Canterbury, New Zealand * Abstract OPEN ACCESS Citation: Tian B, Harrison R, Morton J, DebChoudhury S (2015) Proteomic Analysis of Sauvignon Blanc Grape Skin, Pulp and Seed and Relative Quantification of Pathogenesis-Related Proteins. PLoS ONE 10(6): e0130132. doi:10.1371/ journal.pone.0130132 Academic Editor: Monica Scali, Universita degli Studi di Siena, ITALY Received: February 26, 2015 Accepted: May 18, 2015 Published: June 15, 2015 Copyright: © 2015 Tian et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper. Funding: This research was funded by the New Zealand Winegrowers. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The coauthor, Santanu Deb-choudhury, is employed by a commercial company Agresearch, Lincoln Research Centre. Agresearch, Lincoln Research Centre provided support in the form of salary for author SDC, but did not have any additional role in the study design, data collection and analysis, decision to 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. PLOS ONE | DOI:10.1371/journal.pone.0130132 June 15, 2015 1 / 15 Profiling and Quantification of Proteins in Sauvignon Blanc Grapes publish, or preparation of the manuscript. The specific role of this author is articulated in the ‘author contributions’ section. Competing Interests: The authors declare the following competing interests: This research was funded by the New Zealand Winegrowers. The coauthor, Santanu Deb-choudhury, is employed by a commercial company Agresearch, Lincoln Research Centre. There are no patents, products in development or marketed products to declare. This does not alter the authors' adherence to all the PLoS ONE policies on sharing data and materials. Introduction 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]. Pathogenesis-related proteins are a group of plant proteins induced in pathological or related situations [3]. They were first discovered in tobacco as a result of a hypersensitive reaction to tobacco mosaic virus (TMV) [4]. Pathogenesis-related proteins are typically acidic, of low molecular mass and highly resistant to proteolytic degradation and to low pH values. 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 also been found in grapevine. The two prominent soluble proteins accumulated in grapes during ripening have been identified as chitinases (PR-3 family) and thaumatin-like proteins (PR-5 family) [6,7]. However, in early studies, the β-1,3-glucanases (PR-2 family), a potential indicator of pathogen attack, were not found in grape juice and/or berry extracts [7–10]. 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 [14–16]. Thaumatin-like proteins (TLPs) and chitinases are the two predominent PR protein families present in finished white wine [2,10,17] and they are usually removed by fining with bentonite, a clay material that has a strong affinity for proteins and other larger molecules [18]. However, the addition of bentonite may result in the loss of wine volume (5–20%) as lees and remove important aroma and flavour compounds [19,20]. Recent study showed that bentonite requirement to achieve wine protein stability is strongly correlated with concentration of PR proteins in wine, and specifically has a positive linear correlation with the concentration of chitinases [21]. Thus, a lower concentration of PR proteins in juice and wine, in particular the concentration of chitinases, could reduce the bentonite usag (...truncated)