Aplicación de metodologías proteómicas al estudio de la estructura, modificaciones postraduccionales y diferenciación genética de la proteína faseolina en semilla de la judía común (Phaseolus vulgaris L.)

La judía común (Phaseolus vulgaris L.) es la legumbre de grano más importante para el consumo humano. Las proteínas de reserva de la semilla son la principal fuente de aminoácidos y de nitrógeno reducido que desempeñan un papel clave durante el proceso de la germinación y el crecimiento temprano de la plantas. La faseolina es la principal proteína de reserva de la semilla de la judía común, representando hasta un 40-60% de la proteína total. Décadas de estudios han permitido obtener un conocimiento cada vez más profundo de la base genética, expresión, regulación, estructura, diferenciación genética entre variedades y evolución de la faseolina. Por tanto, se puede considerar que la faseolina representa la proteína de reserva de semilla modelo. La faseolina está codificada por una familia génica localizada en el cromosoma 7. Comprende las subfamilias génicas y que dan lugar a la síntesis de polipéptidos glicosilados del tipo y , respectivamente. El estudio de la diversidad de la faseolina en poblaciones silvestres y cultivadas, mediante el uso de la electroforesis monodimensional, ha permitido la identificación de dos acervos génicos principales de P. vulgaris (mesoamericano y andino) que fue posteriormente confirmada con una diversidad de marcadores de ADN. Sin embargo, los avances recientes en las metodologías proteómicas proporcionan nuevas herramientas para profundizar en el conocimiento de esta compleja familia génica. En este estudio se abordan diversos aspectos sobre la estructura, expresión y diferenciación genética de la faseolina aplicando la electroforesis bidimensional (2-DE) y la espectrometría de masas (MALDI-TOF y MALDI-TOF/TOF MS) en conjunción con otras metodologías proteómicas. La optimización del método de extracción proteica y de los protocolos de la 2-DE ha permitido obtener mapas de alta resolución de la proteína faseolina que suministraron información nueva de gran relevancia. Los resultados del estudio proporcionan la primera evidencia sobre la existencia de isoformas de la faseolina diferencialmente fosforiladas. Además, isoformas con elevados niveles de fosforilación desempeñan un papel clave en la movilización de la proteína faseolina dado que experimentan un proceso de degradación preferente durante la germinación de la semilla. Cabe subrayar que este sería el primer papel funcional asignado a las isoformas fosforiladas detectadas hasta la fecha en las proteínas de reserva de semilla. Por otra parte, el análisis de las distancias proteómicas obtenidas a partir de los patrones 2-DE muestra que la faseolina es un marcador fiable de diferenciación genética y calidad de la semilla en variedades silvestres y cultivadas de los acervos mesoamericano y andino. Por tanto, la faseolina parece ser un marcador de utilidad para la mejora genética de la judía común. Globalmente, el estudio revela que las metodologías proteómicas son una herramienta importante para avanzar en el conocimiento de las complejas proteínas de reserva de semilla

Application of proteomic technologies to assess the quality of raw pork and pork products: an overview from farm-to-fork

The quality assurance of pork meat and products includes the study of factors prior to slaughter such as handling practices, diet and castration, and others during the post-mortem period such as aging, storage, and cooking. The development over the last two decades of high-throughput techniques such as proteomics offer great opportunities to examine the molecular mechanisms and study a priori the proteins in the living pigs and main post-mortem changes and post-translational modifications during the conversion of the muscle into the meat. When the most traditional crossbreeding and rearing strategies to improve pork quality were assessed, the main findings indicate that metabolic pathways early post-mortem were affected. Among the factors, it is well documented that pre-slaughter stress provokes substantial changes in the pork proteome that led to defective meat, and consequently, novel protein biomarkers should be identified and validated. Additionally, modifications in pork proteins had a strong effect on the sensory attributes due to the impact of processing, either physical or chemical. Maillard compounds and protein oxidation should be monitored in order to control proteolysis and volatile compounds. Beyond this, the search of bioactive peptides is becoming a paramount goal of the food and nutraceutical industry. In this regard, peptidomics is a major tool to identify and quantify these peptides with beneficial effects for human health.CYTED | Ref. 119RT0568Axencia Galega de Innovación | Ref. IN607A2019/0

Identification and Mapping of Phosphorylated Isoforms of the Major Storage Protein of Potato Based on Two- Dimensional Electrophoresis

Protein phosphorylation plays a key role in the synthesis and degradation of dry seed storage proteins. In contrast, no evidence for phosphorylation has been reported to date in vegetative storage proteins (VSPs). The patatin multigene family encodes the major VSP of the potato, Solanum tuberosum L. This study addresses for the first time the identification and mapping of phosphorylated patatin forms based on high-resolution two-dimensional electrophoresis (2-DE) profiles. Patatin isoforms from mature tubers of cultivar Kennebec were separated by 2-DE and subsequently identified by tandem mass spectrometry. In-gel identification and mapping of phosphorylated isoforms were performed using the multiplex phosphoprotein-specific staining Pro-Q DPS. We found that phosphorylation is a ubiquitous post-translational protein modification associated with isoforms of patatin. In addition, protein dephosphorylation with hydrogen fluoride-pyridine coupled to 2-DE was used for quantitative profiling of phosphorylated patatin. This experimental approach showed that patatin comprises multiple isoforms with very different phosphorylation level. Thus, phosphorylation rates over isoforms ranged from 4.6 to 52.3%. Overall, the identification and mapping of differentially phosphorylated patatin opens up new exploratory ways to unravel the molecular mechanisms underlying its mobilization along the tuber life cycle

Application of Proteomic Technologies to Assess the Quality of Raw Pork and Pork Products: An Overview from Farm-To-Fork

The quality assurance of pork meat and products includes the study of factors prior to slaughter such as handling practices, diet and castration, and others during the post-mortem period such as aging, storage, and cooking. The development over the last two decades of high-throughput techniques such as proteomics offer great opportunities to examine the molecular mechanisms and study a priori the proteins in the living pigs and main post-mortem changes and post-translational modifications during the conversion of the muscle into the meat. When the most traditional crossbreeding and rearing strategies to improve pork quality were assessed, the main findings indicate that metabolic pathways early post-mortem were affected. Among the factors, it is well documented that pre-slaughter stress provokes substantial changes in the pork proteome that led to defective meat, and consequently, novel protein biomarkers should be identified and validated. Additionally, modifications in pork proteins had a strong effect on the sensory attributes due to the impact of processing, either physical or chemical. Maillard compounds and protein oxidation should be monitored in order to control proteolysis and volatile compounds. Beyond this, the search of bioactive peptides is becoming a paramount goal of the food and nutraceutical industry. In this regard, peptidomics is a major tool to identify and quantify these peptides with beneficial effects for human healthJosé M. Lorenzo and Daniel Franco are members of the HealthyMeat network, funded by CYTED (Programa Iberoamericano de Ciencia y Tecnología para el Desarrollo) (ref. 119RT0568). Our thanks go to GAIN (Axencia Galega de Innovación, Xunta de Galicia, Spain) for supporting this research (grant number IN607A2019/01). Mohammed Gagaoua is grateful to the funding received from Marie Skłodowska-Curie, grant agreement no. 713654S

Patrones electroforéticos en geles bidimensionales de la proteina faseolina en distintas variedades de Phaseolus vulgaris L.

Comunicaciones a congreso

Antioxidant and Antimicrobial Activity of Porcine Liver Hydrolysates Using Flavourzyme

Oxidative stress is implicated in human diseases including cancer or neurodegenerative diseases. On the other hand, lipid and microbial spoilage are the main issues of food degradation. Bioactive peptides with antioxidant and antimicrobial activity could solve both problems and create an opportunity to improve the sustainability of the meat industry. Recently, meat by-products are subject of numerous studies to produce antioxidant peptides, highlighting pork liver as a potential source of hydrolysates. To achieve this purpose, pork liver was digested with Flavourzyme at four reaction times (4, 6, 8, and 10 h) and filtered with cut-offs of 5, 10, and 30-kDa molecular weight. Monitoring hydrolysis with SDS-PAGE showed that the reaction was almost complete. Free amino acid profile exhibited that aliphatic and aromatic amino acids were released in a higher amount at longer reaction times. Heat map analysis demonstrated that a hydrolysis time beyond 6 h, displayed a differential amino acid pattern enabling us to optimize the enzymatic reaction. Antioxidant activity was assessed using ABTS, DPPH, FRAP, and ORAC tests, while antimicrobial assay was carried out against Gram-positive and Gram-negative. ABTS and DPPH values revealed that hydrolysates showed a high antioxidant capacity, as well as an inhibition of growth of Brochothrix thermosphata particularly 30 kDa hydrolysatesAuthors would like to acknowledge to INIA for granting Paula Borrajo with a predoctoral scholarship (grant number CPD2016-0030). José M. Lorenzo is a member of the HealthyMeat network, funded by CYTED (Programa Iberoamericano de Ciencia y Tecnología para el Desarrollo) (ref. 119RT0568). Thanks to GAIN (Axencia Galega de Innovación, Xunta de Galicia, Spain) for supporting this research (grant number IN607A2019/01)S

A proteomic approach to identify biomarkers of foal meat quality: A focus on tenderness, color and intramuscular fat traits

Foal meat is considered a healthy alternative to other meat sources and more environmentally sustainable. However, its quality is highly variable and there is lack of knowledge about the molecular mechanisms underlying its determination. Genotype and diet play a relevant role as the main factors that can allow a control of the final quality and the use of high-throughput analytical methods such as proteomics is a way to achieve this lofty goal. This research aimed to study-two breeds (Burguete and Jaca Navarra) supplemented with two different finishing diets: conventional concentrate and straw (C) vs silage and organic feed (S). The proteomic approach built a library of 294 proteins that were subjected to several statistical and bioinformatic analyses. Burguete breed finished with concentrate produced higher meat quality in terms of tenderness, intramuscular fat and color lightness mainly due to the high abundance of energy metabolic proteins. Tenderness was correlated to myofibrillar proteins (ACTA1, MYBPH, MYL1 and TNNC1) and energy metabolic proteins (ALDOA, CKM, TPI1 and PGMA2). Regarding color, the main pathways were energy metabolism, involving several glycolytic enzymes (ALDOA, PKM, PFKM and CKM). Oxidative stress and response to stress proteins (HSPA1A, SOD2 and PRDX2) were further involved in color variation. Moreover, we revealed that several proteins were related to the intramuscular fat accordingly to the breed. This study proposed several candidate protein biomarkers for foal meat quality that are worthy to evaluate in the futureThis research was funded by Interreg V SUDOE, through OPEN2PRESERVE project, Grant No. SOE2/P5/E0804S

Valorisation of pork by-products to obtain antioxidant and antihypertensive peptides

The porcine liver could be used for the extraction of zinc-protoporphyrin (ZnPP) as a natural red meat pigment. During the autolysis process, porcine liver homogenates was incubated at pH 4.8 and 45 °C under anaerobic conditions to obtain insoluble ZnPP. After incubation, the homogenates were readjusted at pH 4.8, and at pH 7.5 before being centrifuged at 5500 × g for 20 min at 4 °C and the resulting supernatant were compared with the obtained at pH 4.8 at the beginning of the incubation. The molecular weight distributions of the porcine liver fractions at both pHs were very similar, however, eight essential amino acids were more abundant in fractions obtained at pH 4.8. Regarding the ORAC assay, porcine liver protein fraction at pH 4.8 showed the highest antioxidant capacity but antihypertensive inhibition was similar for both pHs. Peptides with strong bioactivity potential from aldehyde dehydrogenase, lactoylglutathione lyase, SEC14-like protein 3 and others were identified. The findings have demonstrated the potential of the porcine liver to extract natural pigments and bioactive peptides.info:eu-repo/semantics/publishedVersio

The first evidence of global meat phosphoproteome changes in response to pre-slaughter stress

Pre-slaughter stress (PSS) impairs animal welfare and meat quality. Dark, firm and dry (DFD) are terms used to designate poor quality meats induced by PSS. Protein phosphorylation can be a potentially significant mechanism to explain rapid and multiple physiological and biochemical changes linked to PSS-dependent muscle-to-meat conversion. However, the role of reversible phosphorylation in the response to PSS is still little known. In this study, we report a comparative phosphoproteomic analysis of DFD and normal meats at 24 h post-mortem from the longissimus thoracis (LT) bovine muscle of male calves of the Rubia Gallega breed. For this purpose, two-dimensional gel electrophoresis (2-DE), in-gel multiplex identification of phosphoproteins with PRO-Q Diamond phosphoprotein-specific stain, tandem (MALDI-TOF/TOF) mass spectrometry (MS), novel quantitative phosphoproteomic statistics and bioinformatic tools were usedMass spectrometry analysis, writing of the manuscript and article-processing charges were supported by grant RTA 2014–00034-C04 from the Instituto Nacional de Investigación y Tecnología Agraria (INIA, Spain). Meat samples were obtained by a project FEADER 2010–04 (Consellería de Medio Rural of Xunta de Galicia, Spain)S

Proteomic Advances in Milk and Dairy Products

Proteomics is a new area of study that in recent decades has provided great advances in the field of medicine. However, its enormous potential for the study of proteomes makes it also applicable to other areas of science. Milk is a highly heterogeneous and complex fluid, where there are numerous genetic variants and isoforms with post-translational modifications (PTMs). Due to the vast number of proteins and peptides existing in its matrix, proteomics is presented as a powerful tool for the characterization of milk samples and their products. The technology developed to date for the separation and characterization of the milk proteome, such as two-dimensional gel electrophoresis (2DE) technology and especially mass spectrometry (MS) have allowed an exhaustive characterization of the proteins and peptides present in milk and dairy products with enormous applications in the industry for the control of fundamental parameters, such as microbiological safety, the guarantee of authenticity, or the control of the transformations carried out, aimed to increase the quality of the final productThis research was funded by GAIN (Axencia Galega de Innovación, Xunta de Galicia, Spain), grant number IN607A2019/01S