Journal of Proteomics 211 (2020) 103530

Journal of Proteomics 211 (2020) 103530 Contents lists available at ScienceDirect Journal of Proteomics journal homepage: www.elsevier.com/locate/jprot Qualitative proteomic comparison of metabolic and CM-like protein fractions in old and modern wheat Italian genotypes by a shotgun approach T ⁎ Antonella Di Francescoa, Rosaria Salettia, , Vincenzo Cunsoloa, Birte Svenssonb, Vera Muccillia, Pasquale De Vitac, Salvatore Fotia a Laboratory of Organic Mass Spectrometry, Department of Chemical Sciences, University of Catania, Viale A. Doria 6, 95125 Catania, Italy b Department of Biotechnology and Bioengineering, Technical University of Denmark, Søltofts Plads, Building 224, Kgs. Lyngby DK-2800, Denmark c CREA Research Centre for Cereal and Industrial Crops (CREA-CI), S.S. 673 km 25.200, 71122 Foggia, Italy ARTICLE INFO ABSTRACT Keywords: The close relationship between diet and health is generally recognized and the growing wellness and con- Old and modern wheat genotypes sciousness, especially in developed countries, have led to increasing interest for old wheat genotypes, based on High resolution mass spectrometry perceived health benefits. Although nutritional comparison between old and modern wheat varieties is still Proteome analysis controversial, it is generally accepted that old wheat genotypes remained unchanged over the last hundred years. Proteins By contrast, modern wheat genotypes are derived by modification of old wheats during the so-called “Green- Allergens Revolution” in the second half of the 20th century focusing on obtaining properties in terms of higher grain Food and nutrition yield. The present work reports the first comprehensive proteomic profiling and qualitative comparison at the molecular level of metabolic and Chloroform-Methanol (CM)-like protein fractions extracted from mature ker- nels of two old Sicilian durum wheat landraces, Russello and Timilia Reste Bianche, and Simeto, an improved durum wheat variety widespread in Italy and other Mediterranean countries and chosen as representative of the most widely commercial cultivars. The results obtained reveal that metabolic and CM-like protein fractions of old and modern genotypes present remarkably high similarity with only minor differences. This leads to the conclusion that from a food and nutritional perspective there is a substantial equivalence of the protein composition of the old and modern cultivars. Data are available via ProteomeXchange with identifier PXD014449. Biological significance: In recent years consumers have shown growing interest in the old wheat genotypes, which are generally perceived more “natural” and healthier than modern ones. However, comparison of nutritional value for modern and old wheat varieties is still controversial suggesting further studies. In particular proteome analysis of old and modern wheat genotypes is currently ongoing with particular focus on gluten proteins, whereas the metabolic protein fraction has not yet been investigated. In the present study, we conducted a comprehensive proteomic profile and qualitative comparison at the molecular level of metabolic and Chloroform-Methanol (CM)-like protein fractions of the old Sicilian landraces Russello and Timilia Reste Bianche and the modern cultivar Simeto by applying a shotgun approach. The results reveal that the metabolic and CM- like protein fractions of old and modern genotypes are remarkably similar with only minor differences, leading to the conclusion that from a food and nutritional perspective there is a substantial equivalence of these cultivars. These results may contribute to improved understanding of the relationship between protein profiles of old wheat genotypes and their potential benefits for human consumption. 1. Introduction characterization of wheat proteins by MS-based methods [5–7]. The aim of these studies is not limited to characterization of the sequence of Wheat is one of the most important cereals for mankind. Wheat is kernel proteins or to proteomics investigation of cultivars [8–13], but not only a source of calories for the human diet but it also provides also include efforts to better understand the role of these components in essential amino acids, minerals, vitamins, and bioactive compounds the gluten matrix [14–17]. [1–4]. For this reason, wheat has been extensively studied over the In spite of its central role for human nutrition, wheat may also cause years. In particular, most of these studies focused on the several adverse reactions and disorders, such as immunoglobulin E ⁎ Corresponding author. E-mail address: [email protected] (R. Saletti). https://doi.org/10.1016/j.jprot.2019.103530 Received 3 May 2019; Received in revised form 3 September 2019; Accepted 17 September 2019 Available online 16 October 2019 1874-3919/ © 2019 Elsevier B.V. All rights reserved. A. Di Francesco, et al. Journal of Proteomics 211 (2020) 103530 (IgE) mediated allergies (celiac disease, CD), which requires a lifelong 2. Materials and methods gluten free diet (GFD) [18], and a less well-defined condition classified as non-celiac wheat sensitivity (NCWS) [19–23]. Therefore, the devel- 2.1. Chemicals opment and characterization of transgenic low-gliadin wheat lines suitable for CD is ongoing [24], even though this will be difficult to All chemicals were of the highest purity commercially available and achieve. The metabolic and CM-like protein fractions include other were used without further purification. KCl, K2HPO4, acetone, me- allergens, promoting IgE mediated reactions. A typical example of thanol, acetic acid and Tris-HCl were purchased from Carlo Erba wheat allergy is Baker's asthma [25,26]. The most commonly re- (Milan, Italy). Formic Acid (FA), Protease Inhibitor Cocktail, EDTA, cognized allergens are the α-amylase/trypsin inhibitor subunits [27], ammonium bicarbonate, ammonium acetate, dithiothreitol (DTT), io- but recently several other proteins have been linked to wheat allergy; in doacetamide (IAA) were obtained from Aldrich (St. Louis, Missouri, particular, lipid transfer proteins (LTPs) [28,29], peroxidase [26], USA). Modified porcine trypsin was purchased from Promega (Madison, thioredoxins [26], serine proteinase inhibitors (serpins) [26,30], thau- WI, USA). Water and acetonitrile (ACN) (OPTIMA® LC/MS grade) for matin-like protein [26], acyl-CoA oxidase [26], fructose-bisphosphate LC/MS analyses were purchased from Fisher Scientific (Milan, Italy). aldolase [26], triosephosphate isomerase [26], and glyceraldehyde-3- LDS sample buffer, Mark12™ Unstained Standards and SimplyBlue™ phosphate dehydrogenase [26]. Safe Stain were obtained from Invitrogen™ (Life Technologies™, Paisley, It has been suggested that old wheat species may have a healthier UK). and superior nutritional profile than modern wheat species, by being richer in vitamins, minerals and nutraceutical compounds [31–33]. 2.2. Samples collection and treatment Although there is no precise clarification, it is generally accepted that old wheat genotypes have remained unchanged over the last hundred Three biological replicates of Russello, Timilia RB and Simeto were years. By contrast, modern wheat cultivars were generated during the provided from CREA-CI. The genetic materials were sowed at Foggia, so-called “Green-Revolution” during the second half of the 20th cen- during the 2010–11 growing season, following a randomized block tury. The most common old wheat species commercially available are design with three replicates. Grain samples were harvested and the einkorn (T. monococcum L. ssp. monococcum), Emmer (T. turgidum L. ssp. flours were stored at 4 °C. Flours (200 mg) were suspended in 2 mL cold dicoccum), Khorasan (T. turgidum ssp. turanicum) and spelt (T. aestivum (4 °C) extraction solution (50 mM Tris-HCl, 100 mM KCl, 5 mM EDTA, L. ssp. spelta)[34]. In addition, there are several old genotypes of both Protease Inhibitor Cocktail, pH 7.8) in order to obtain the metabolic T. aestivum and T. durum, cultivated from the mid-1800s, to the be- and Chloroform-Methanol (CM)-like proteins. The solution was in- ginning of the 20th century (before the “Green Revolution) including cubated on ice (5 min) with intermittent mixing and centrifuged cultivars of durum wheat such as Russello, Senatore Cappelli, Timilia or (13,523g, 15 min, 4 °C). The supernatant was collected and added five Tumminia, and bread wheat such as Gentil Rosso, Maiorca, Sieve, Solina, volumes of 0.1 M ammonium acetate in methanol. Following incuba- and Verna [33]. In the literature, the nutritional comparison between tion overnight at −20 °C, the solution was centrifuged (30g, 15 min, old and modern wheat varieties is still debated, also due to the limited R.T.). The pellet (containing the metabolic proteins) was collected and number of genotypes investigated, and it has been suggested that fur- rinsed in 3 mL 0.1 M ammonium bicarbonate, pH 8.2. Proteins in the ther studies are urgently required [35]. Generally, it is reported that the supernatant (CM-like proteins) were precipitated by addition of four health benefit of old grains is not related to a single compound but to volumes of cold acetone, kept overnight at −20 °C and subsequently their general composition. In particular, information on the proteomic centrifuged (30g, 15 min, R.T.). Finally, the pellet containing CM-like comparison of these genotypes with modern wheat varieties is very proteins was rinsed by 1.5 mL 0.1 M ammonium bicarbonate, pH 8.2 scant [36]. Recently, a set of old and modern Italian tetraploid wheat [39]. The protein concentration for each extract

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