Determination of color, antioxidant activity and phenolic profile of different fruit tissue of Spanish ‘Verde Doncella’ apple cultivar

27 Pag., 1 Fig., 4 Tab. Accepted author version posted online 16 January 2014. Published online: 14 Jul 2014. This is an Author's Accepted Manuscript of an article published in International Journal of Food Properties, published online 16 Jan 2014, copyright Taylor & Francis, available online at: http://www.tandfonline.com/10.1080/10942912.2013.792829The polyphenolic profile and antioxidant activity of peel, pomace, and juice of ‘Verde Doncella’, a Spanish apple cultivar is presented. Phenolic profile of the worldwide cultivated, ‘Red Delicious’ cultivar was used for comparison. Flavanols, hydroxycinamic acids, flavonols, phloridzin, procyanidin B2, and gallic acid were quantified by HPLC. Larger concentrations of polyphenolics were found in the peel, which is in agreement with the Total Phenolic Content (TPC) and antioxidant activity (FRAP) values. ‘Verde Doncella’ expressed lower concentrations of flavanols and quercetin derivates in peel, pomace, and juice when compared to ‘Red Delicious’. ‘Verde Doncella’ was richer in p-coumaric acid and procyanidn B2 in the peel.This study was supported by the projects AGL2009-59 08501 (Programa Nacional de Proyectos de Investigación Fundamental), co-financed by the European Social Fund and by the Departamento de Ciencia, Tecnología y Universidad del Gobierno de Aragón, Research Group “Foods from Plant Origin”. Fellowship funding JAE-Predoc/CSIC was supported by the Ministerio de Ciencia e Innovación (MICCIM).Peer reviewe

Identification of bitter pit markers in Malus domestica

Bitter pit is a physiological disorder occurring in apple, pear, and quince and has been associated with calcium uptake or lack thereof. Studied for over a century, little is known regarding the development of bitter pit, what triggers its occurrence, and why there are no completely effective preventative treatments to reduce fruit loss. In the present study, Malus domestica proteins were extracted, injected, and analyzed on a LC Q Exactive mass spectrometer. More than 1500 proteins were identified between bitter pit and healthy samples. To establish quality markers of bitter pit, proteins were filtered using p<0.05 (95 % significance). A total of 170 proteins were identified as quality markers for bitter pit (Whether significantly greater or lower). This study is first to report the bitter pit protein profile in Malus domestica and identify significantly different markers between bitter pit and healthy samples that indicate the occurrence of bitter pit.Peer Reviewe

Polyphenolic and Proteomic Characterization of Malus domestica Fruit Affected by Bitter Pit

221 Pags.Bitter pit is a physiological disorder occurring in apple, pear, and quince whose symptoms are known to develop in storage, appearing several weeks to months after harvest. This disorder causes commercial losses for apple growers as affected fruits are declined or achieve lower prices in the market. Uneven calcium distribution in fruit tissue has been thought to be one of several factors that cause bitter pit. Despite bitter pit has been studied for more than a century, the mechanisms involved in its development are still not well understood. To date, most of the research carried out on bitter pit has been focused on Ca2+ deficiency. However, little to no attention has been paid to the expression of other important metabolites indicative of cell status. Phenolic compounds and proteins are end products of numerous cellular processes occurring in the biological systems as a result of natural defensive reactions against stress and diseases. In the quest to understand bitter pit, the study of these chemical “fingerprints” cannot be avoided, as they can help to better understand the biochemical mechanisms involved in the development of this disorder. Under this premise, this research has addressed the analysis and comparison of phenolic compounds and proteins present in healthy and bitter pit affected tissues of apple (Malus domestica Borkh). For this purpose, several approaches and techniques have been employed. The analysis of phenolic compounds and antioxidant activity in healthy apples was first carried in order to optimize the methodology (Chapter 1). Even if most of phenolic families in apple could be successfully identified, the methodology employed, high performance liquid chromatography coupled to a UV-DAD detector, presented some limitations to accurately identify all the eluted peaks. As a result, an alternative methodology based on mass spectra detection of target phenolic compounds was employed to compare the phenolic profile of healthy and bitter pit tissues (Chapter 2). With respect to the study of proteins, the identification of an 18 kDa protein suggested as a potential bitter pit marker by former group studies was first carried out (Chapter 3). The characterization of additional proteins overly expressed in bitter pit tissues was next addressed by means of most sophisticated proteomic-based analytical strategies (Chapters 4 & 5). Results suggest that down-regulation in the expression of major phenolic compounds and low antioxidant activities were associated with the presence of bitter pit disorder, which may suggest that oxidative activity accompanies bitter pit disorder development. Bitter pit disorder also induced a deep change in the protein profile of affected tissues, expressing a variety of pathogenesis-related (PR) proteins from several families, including Mal d 1 and Mal d 2, two major allergens. Other proteins with diverse cell functions such as tissue desiccation, mitochondrial carrying or protein binding among others were found to be upregulated in bitter pit tissues.Peer reviewe

Identification and quantification of proteins in Malus domestica affected by bitter pit

Bitter pit is a physiological disorder that occurs in apple, pear, and quince and has been associated with calcium uptake or lack thereof. Although bitter pit has been studied for over a century, there is still not enough knowledge about bitter pit and why there are no completely effective preventive treatments to reduce fruit loss. In a previous group publication (Val et al. 2006), it was conjectured after SDS-PAGE that an unknown 18 kDa protein might contribute to bitter pito The objective of the present study was to identify this 18 kDa protein and get more extensive data on the proteomic changes associated to bitter pit using the latest mass spectrometry-based proteomics. Healthy and bitter pit fruit samples (Malus domesica 'Reinette gris du Canada' and Malus domestica 'Golden Smoothee') were collected near Zaragoza (Aragón, Spain). Following phenol extraction, ten µg protein were allowed to run on SDS-PAGE, trypsin digested, and digestion products were analyzed on a Q-Exactive mass spectrometer (Thermo Scientific). Proteins were identified with X!Tandem pipeline software (http://pappso.inra.fr/) and relative quantification was performed by spectral counting. More than two hundred proteins were identified in the range of 12-24 kDa. We focused on 35 proteins that varied significantly between the two conditions (bitter pit vs. healthy) being over/under expressed. There were 26 and 22 bitter pit proteins ('Reinette gris du Canada' and 'Smoothee Golden Delicious,' respectively) detected with at least 50% greater abundance when compared to their respective healthy counterparts. Among these proteins, 14 found in both cultivars, were identified as Pathogenesis-related protein Bet v 1, a major allergen found in trees within the order Fagales. In both apple cultivars, 2 proteins were identified as thaumatin-like protein (TLP), a group of proteins responsible for several fruit allergies. Glutathione S-transferase, linked to protein binding and heat shock transcriptional factors (Hsfs) in Malus, was abundantly detected in bitter pit samples for both cultivars. Several proteins identified near or at 18-kDa, are related to Malus domestica' response to stress, desiccation, and increased protein binding. Considerable differences were found in allergen concentrations between bitter pit and healthy samples, suggesting an increased allergen risk for consumers who ingest bitter pit affected fruit.Peer Reviewe

Identification of Polypeptides in Apple (Malus x domestica Borkh.) that may assist in determining plant stress related diseases

6 Pags., 1 Fig. Esta contribución científica en forma de capítulo de libro fue originalmente presentada por los autores en el X Simposio Nacional y VII Ibérico sobre Maduración y Postcosecha de Frutas y Hortalizas (Lleida, 1-4 de Octubre 2012).The occurrence of nutrient-related disorders in apple fruit, such as bitter pit, cork and Jonathan spot, to name a few, are increasing problems of great importance for the fruit industry. Although bitter pit has been recognized and characterized for more than a century, there is little information related to the development of physiological disorders in fruit that trigger their occurrence. In a previous group study, a novel 18 kDa protein was found in bitter pit affected tissues, suggesting this protein band may be an indicator/marker for bitter pit disease in apples. No comparison was made between bitter pit diseased fruit and other fruit showing symptoms of another disease. The 18 kDa protein has been reported to contain Mal d 1, the major apple allergen that is believed to be pathogen and stress induced. Polypeptides from apple cultivar 'Golden Delicious' affected with bitter pit, and apple cultivar ‘Transparente Blanco’ affected with watercore were analysed with one-dimensional denaturing sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) to determine protein bands unique to each disease. Results indicate the 18 kDa protein was found not only in bitter pit affected apple tissues but also in watercore tissues. These findings do not dismiss the possibility that the 18 kDa protein band is an indicator for bitter pit, but potentially broadens the range the 18 kDa band may have as an indicator for disease or nutrient deficiency caused by plant stress.This work was financed by MINECO (Spanish Ministry of Economy and Competitivity) under the projectAGL2009-08501.Peer reviewe

Identification of bitter pit protein markers in Malus domestica using differential in-gel electrophoresis (DIGE) and LC–MS/MS

36 Pags.- 7 Figs.- 3 Tabls. The definitive version is available at: http://www.sciencedirect.com/science/journal/09255214Bitter pit is a physiological disorder that occurs in apple, pear and quince and has long been associated with calcium uptake or lack thereof. In the present study, pooled biological Malus domestica proteins were collected from healthy and naturally occurring bitter pit fruit. Protein samples (bitter pit and healthy) were analyzed with differential in-gel electrophoresis (DIGE) and SameSpots software was used to compare gel spots by intensity. Identified spots (p < 0.05) were spot picked and trypsin digested. Peptides were separated by liquid chromatography (LC) and submitted to LTQ-Orbitrap mass spectrometer to infer protein identification. A total of 200 ± 5 protein spots were detected, 41 spots classified as having p < 0.05 and were successfully identified by their peptide sequence listed in an online M. domestica database. Thirteen spots were identified as having p < 0.05 and a minimum 2-fold change. Several pathogenesis-related (PR) proteins belonging to three PR families, PR-5 (10.3-fold), PR-8/chitinase (35.5-fold) and PR-10 (6.8-fold) were upregulated in bitter pit sampled tissues. Three proteins involved in several metabolic processes including ethylene biosynthesis (3.3-fold), glycosyltransferase reactions in metabolism (2.3-fold) and metal biding (11.1-fold) were found to be overly expressed in healthy sampled tissues. This research provides a significant advance in the knowledge of protein expression alterations occurring in bitter pit in comparison with sound tissues.This study was supported by the projects AGL2009-59 08501 (Programa Nacional de Proyectos de Investigación Fundamental), co-financed by the European Social Fund. Fellowship funding JAE-Predoc/CSIC was supported by the Ministerio de Economía y Competitividad (MINECO). ProteoRed is part of the PRB2-ISCIII, supported by grant PT13/0001.Peer reviewe

High Performance Anion Exchange and Hydrophilic Interaction Liquid Chromatography Approaches for Comprehensive Mass Spectrometry-Based Characterization of the N‑Glycome of a Recombinant Human Erythropoietin

Comprehensive characterization of the N-glycome of a therapeutic is challenging because glycans may harbor numerous modifications (e.g., phosphorylation, sulfation, sialic acids with possible O-acetylation). The current report presents a comparison of two chromatographic platforms for the comprehensive characterization of a recombinant human erythropoietin (rhEPO) N-glycome. The two platforms include a common workflow based on 2-AB-derivatization and hydrophilic interaction chromatography (HILIC) and a native N-linked glycan workflow employing high performance anion exchange (HPAE) chromatography. Both platforms were coupled to an Orbitrap mass spectrometer, and data dependent HCD fragmentation allowed confident structural elucidation of the glycans. Each platform identified glycans not revealed by the other, and both exhibited strengths and weaknesses. The reductive amination based HILIC workflow provided better throughput and sensitivity, had good isomer resolution, and revealed the presence of O-acetylated sialic acids. However, it exhibited poor performance toward phosphorylated glycans and did not reveal the presence of sulfated glycans. Furthermore, reductive amination introduced dehydration artifacts and modified the glycosylation profile in the rhEPO glycome. Conversely, HPAE provided unbiased charge classification (sialylation levels), improved isomer resolution, and revealed multiple phosphorylated and sulfated structures, but delivered lower throughput, had artifact peaks due to epimer formation, and loss of sialic acid O-acetylation. The MS² based identification of phosphorylated and sulfated glycans was not possible in HILIC mode due to their poor solubility caused by the high acetonitrile concentrations employed at the beginning of the gradient. After analyzing the glycome by both approaches and determining the glycans present, a glycan library was created for site specific glycopeptide analyses. Glycopeptide analyses confirmed all the compositions annotated by the combined use of 2-AB- and native glycan workflows and provided site specific location of the glycans. These two platforms were complementary and in combination delivered a more thorough and comprehensive characterization of the rhEPO N-glycome, supporting regulatory conformance for the pharmaceutical industry