Giovanni Renzone

A proteomic characterization of water buffalo milk fractions describing PTM of major species and the identification of minor components involved in nutrient delivery and defense against pathogens

Water buffalo has been studied in relation to the exclusive use of its milk for the manufacture of high‐quality dairy products. Buffalo milk presents physicochemical features different from that of other ruminant species, such as a higher content of fatty acids and proteins. We report here a detailed proteomic analysis of buffalo skim milk, whey and milk fat globule membrane fractions. Notwithstanding the poor information available on buffalo genome, identification of protein isoforms corresponding to 72 genes was achieved by a combined approach based on 2‐DE/MALDI‐TOF PMF and 1‐DE/μLC‐ESI‐IT‐MS‐MS. Major protein components, i.e. αSl‐, αS2‐, β‐, κ‐caseins, α‐lactalbumin and β‐lactoglobulin, were characterized for PTM, providing a scientific basis to coagulation/cheese making processes used in dairy productions. Minor proteins detected emphasized the multiple functions of milk, which besides affording nutrition to the newborn through its major components, also promotes development and digestive tract protection in the neonate, and ensures optimal mammary gland function in the mother. Defense against pathogens is guaranteed by an arsenal of antimicrobial/immunomodulatory proteins, which are directly released in milk or occur on the surface of secreted milk‐lipid droplets. Proteins associated with cell signaling or membrane/protein trafficking functions were also identified, providing putative insights into major secretory pathways in mammary epithelial cells.

Modern proteomic methodologies for the characterization of lactosylation protein targets in milk

Heat treatment of milk induces the Maillard reaction between lactose and proteins; in this context, β‐lactoglobulin and α‐lactalbumin adducts have been used as markers to monitor milk quality. Since some milk proteins have been reported as essential for the delivery of microelements and, being resistant against proteolysis in the gastrointestinal tract, also contributing to the acquired immune response against pathogens and the stimulation of cellular proliferation, it is crucial to systematically determine the milk subproteome affected by the Maillard reaction for a careful evaluation of aliment functional properties. This is more important when milk is the unique nutritional source, as in infant diet. To this purpose, a combination of proteomic procedures based on analyte capture by combinatorial peptide ligand libraries, selective trapping of lactosylated peptides by m‐aminophenylboronic acid‐agarose chromatography and collision‐induced dissociation and electron transfer dissociation MS was used for systematic identification of the lactosylated proteins in milk samples subjected to different thermal treatments. An exhaustive modification of proteins was observed in milk powdered preparations for infant nutrition. Globally, this approach allowed the identification of 271 non‐redundant modification sites in 33 milk proteins, which also included low‐abundance components involved in nutrient delivery, defence response against virus/microorganisms and cellular proliferative events. A comparison of the modified peptide identification percentages resulting from electron transfer dissociation or collision‐induced dissociation fragmentation spectra confirmed the first activation mode as most advantageous for the analysis of lactosylated proteins. Nutritional, biological and toxicological consequences of these findings are discussed on the basis of the recent literature on this subject, emphasizing their impact on newborn diet.

Leaf proteome analysis of transgenic plants expressing antiviral antibodies

The expression of exogenous antibodies in plant is an effective strategy to confer protection against viral infection or to produce molecules with pharmaceutical interest. However, the acceptance of the transgenic technology to obtain self-protecting plants depends on the assessment of their substantial equivalence compared to non-modified crops with an established history of safe use. In fact, the possibility exists that the introduction of transgenes in plants may alter expression of endogenous genes and/or normal production of metabolites. In this study, we investigated whether the expression in plant of recombinant antibodies directed against viral proteins may influence the host leaf proteome. Two transgenic plant models, generated by Agrobacterium tumefaciens-mediated transformation, were analyzed for this purpose, namely, Lycopersicon esculentum cv. MicroTom and Nicotiana benthamiana, expressing recombinant antibodies against cucumber mosaic virus and tomato spotted wilt virus, respectively. To obtain a significant representation of plant proteomes, optimized extraction procedures have been devised for each plant species. The proteome repertoire of antibody-expressing and control plants was compared by 2-DE associated to DIGE technology. Among the 2000 spots detected within the gels, about 10 resulted differentially expressed in each transgenic model and were identified by MALDI-TOF PMF and muLC-ESI-IT-MS/MS procedures. Protein variations were restricted to a limited number of defined differences with an average ratio below 2.4. Most of the differentially expressed proteins were related to photosynthesis or defense function. The overall results suggest that the expression of recombinant antibodies in both systems does not significantly alter the leaf proteomic profile, contributing to assess the biosafety of resistant plants expressing antiviral antibodies.

Molecular interactions between the olive and the fruit fly Bactrocera oleae

BackgroundThe fruit fly Bactrocera oleae is the primary biotic stressor of cultivated olives, causing direct and indirect damages that significantly reduce both the yield and the quality of olive oil. To study the olive-B. oleae interaction, we conducted transcriptomic and proteomic investigations of the molecular response of the drupe. The identifications of genes and proteins involved in the fruit response were performed using a Suppression Subtractive Hybridisation technique and a combined bi-dimensional electrophoresis/nanoLC-ESI-LIT-MS/MS approach, respectively.ResultsWe identified 196 ESTs and 26 protein spots as differentially expressed in olives with larval feeding tunnels. A bioinformatic analysis of the identified non-redundant EST and protein collection indicated that different molecular processes were affected, such as stress response, phytohormone signalling, transcriptional control and primary metabolism, and that a considerable proportion of the ESTs could not be classified. The altered expression of 20 transcripts was also analysed by real-time PCR, and the most striking differences were further confirmed in the fruit of a different olive variety. We also cloned the full-length coding sequences of two genes, Oe-chitinase I and Oe-PR27, and showed that these are wound-inducible genes and activated by B. oleae punctures.ConclusionsThis study represents the first report that reveals the molecular players and signalling pathways involved in the interaction between the olive fruit and its most damaging biotic stressor. Drupe response is complex, involving genes and proteins involved in photosynthesis as well as in the production of ROS, the activation of different stress response pathways and the production of compounds involved in direct defence against phytophagous larvae. Among the latter, trypsin inhibitors should play a major role in drupe resistance reaction.

Non-enzymatic glycation and glycoxidation protein products in foods and diseases: an interconnected, complex scenario fully open to innovative proteomic studies.

The Maillard reaction includes a complex network of processes affecting food and biopharmaceutical products; it also occurs in living organisms and has been strictly related to cell aging, to the pathogenesis of several (chronic) diseases, such as diabetes, uremia, cataract, liver cirrhosis and various neurodegenerative pathologies, as well as to peritoneal dialysis treatment. Dozens of compounds are involved in this process, among which a number of protein-adducted derivatives that have been simplistically defined as early, intermediate and advanced glycation end-products. In the last decade, various bottom-up proteomic approaches have been successfully used for the identification of glycation/glycoxidation protein targets as well as for the characterization of the corresponding adducts, including assignment of the modified amino acids. This article provides an updated overview of the mass spectrometry-based procedures developed to this purpose, emphasizing their partial limits with respect to current proteomic approaches for the analysis of other post-translational modifications. These limitations are mainly related to the concomitant sheer diversity, chemical complexity, and variable abundance of the various derivatives to be characterized. Some challenges to scientists are finally proposed for future proteomic investigations to solve main drawbacks in this research field.

Transcriptomic and proteomic analysis of a compatible tomato-aphid interaction reveals a predominant salicylic acid-dependent plant response

BackgroundAphids are among the most destructive pests in temperate climates, causing significant damage on several crops including tomato. We carried out a transcriptomic and proteomic study to get insights into the molecular mechanisms and dynamics of the tomato response to the Macrosyphum euphorbiae aphid.ResultsThe time course analysis of aphid infestation indicated a complex, dynamic pattern of gene expression. Several biological functions were affected and genes related to the stress and defence response were the most represented. The Gene Ontology categories of the differentially expressed genes (899) and identified proteins (57) indicated that the tomato response is characterized by an increased oxidative stress accompanied by the production of proteins involved in the detoxification of oxygen radicals. Aphids elicit a defense reaction based on the cross-communication of different hormone-related signaling pathways such as those related to the salicylic acid (SA), jasmonic acid (JA), ethylene and brassinosteroids. Among them, the SA-signaling pathway and stress-responsive SA-dependent genes play a dominant role. Furthermore, tomato response is characterized by a reduced accumulation of photosynthetic proteins and a modification of the expression of various cell wall related genes.ConclusionsOur work allowed a more comprehensive understanding of the signaling events and the defense dynamics of the tomato response to aphids in a compatible interaction and, based on experimental data, a model of the tomato–aphid molecular interaction was proposed. Considering the rapid advancement of tomato genomics, this information will be important for the development of new protection strategies.

Proteomic analysis of stress-responsive proteins in Arabidopsis thaliana rosette leaves.

Plants, as sessile organisms, are continuously exposed to temperature changes in the environment. Low and high temperature stresses have a great impact on agricultural productivity, since they significantly alter plant metabolism and physiology. Plant response to temperature stress is a quantitative character, being influenced by the degree of stress, time of exposure, as well as plant adaptation ability; it involves profound cellular changes at the proteomic level. We describe here the quantitative variations of the protein repertoire of Arabidopsis thaliana rosette leaves after exposing seedlings to either short-term cold or heat temperature stress. A proteomic approach, based on two-dimensional electrophoresis and MALDI-TOF peptide mass fingerprinting and/or nanoLC-ESI-LIT-MS/MS experiments, was used for this purpose. The comparison of the resulting proteomic maps highlighted proteins showing quantitative variations induced by temperature treatments. Thirty-eight protein spots exhibited significant quantitative changes under at least one stress condition. Identified, differentially-represented proteins belong to two main broad functional groups, namely energy production/carbon metabolism and response to abiotic and oxidative stresses. The role of the identified proteins is discussed here in relation to plant adaptation to cold or heat stresses. Our results suggest a significant overlapping of the responses to opposite temperature extremes.

Response to biotic and oxidative stress in Arabidopsis thaliana: Analysis of variably phosphorylated proteins

Protein phosphorylation plays a pivotal role in the regulation of many cellular events; increasing evidences indicate that this post-translational modification is involved in plant response to various abiotic and biotic stresses. Since phosphorylated proteins may be present at low abundance, enrichment methods are generally required for their analysis. We here describe the quantitative changes of phosphoproteins present in Arabidopsis thaliana leaves after challenging with elicitors or treatments mimicking biotic stresses, which stimulate basal resistance responses, or oxidative stress. Phosphoproteins from elicited and control plants were enriched by means of metal oxide affinity chromatography and resolved by 2D electrophoresis. A comparison of the resulting proteomic maps highlighted phosphoproteins showing quantitative variations induced by elicitor treatment; these components were identified by MALDI-TOF peptide mass fingerprinting and/or nanoLC-ESI-LIT-MS/MS experiments. In total, 97 differential spots, representing 75 unique candidate phosphoproteins, were characterized. They are representative of different protein functional groups, such as energy and carbon metabolism, response to oxidative and abiotic stresses, defense, protein synthesis, RNA processing and cell signaling. Ascertained protein phosphorylation found a positive confirmation in available Arabidopsis phosphoproteome database. The role of each identified phosphoprotein is here discussed in relation to plant defense mechanisms. Our results suggest a partial overlapping of the responses to different treatments, as well as a communication with key cellular functions by imposed stresses.

Proteomic characterization of intermediate and advanced glycation end-products in commercial milk samples.

UNLABELLED The Maillard reaction consists of a number of chemical processes affecting the structure of the proteins present in foods. We previously accomplished the proteomic characterization of the lactosylation targets in commercial milk samples. Although characterizing the early modification derivatives, this analysis did not describe the corresponding advanced glycation end-products (AGEs), which may be formed from the further oxidation of former ones or by reaction of oxidized sugars with proteins, when high temperatures are exploited. To fill this gap, we have used combined proteomic procedures for the systematic characterization of the lactosylated and AGE-containing proteins from the soluble and milk fat globule membrane fraction of various milk products. Besides to confirm all lactulosyl-lysines described previously, 40 novel lactosylation sites were identified. More importantly, 308 additional intermediate and advanced glyco-oxidation derivatives (including cross-linking adducts) were characterized in 31 proteins, providing the widest qualitative inventory of modified species ascertained in commercial milk samples so far. Amadori adducts with glucose/galactose, their dehydration products, carboxymethyllysine and glyoxal-, 3-deoxyglucosone/3-deoxygalactosone- and 3-deoxylactosone-derived dihydroxyimidazolines and/or hemiaminals were the most frequent derivatives observed. Depending on thermal treatment, a variable number of modification sites was identified within each protein; their number increased with harder food processing conditions. Among the modified proteins, species involved in assisting the delivery of nutrients, defense response against pathogens and cellular proliferation/differentiation were highly affected by AGE formation. This may lead to a progressive decrease of the milk nutritional value, as it reduces the protein functional properties, abates the bioavailability of the essential amino acids and eventually affects food digestibility. These aspects are of particular importance in products intended for infant diet, such as milk powders and infant formulas. BIOLOGICAL SIGNIFICANCE We used combined shotgun proteomic procedures for the systematic characterization of intermediate and advanced glycoxidation protein products in various raw and commercial milk samples. Several hundreds of modified species were characterized as deriving from 31 milk proteins, providing the widest qualitative inventory of assigned components in this fluid. Amadori adducts with glucose/galactose, their dehydration products, carboxymethyl-lysine, and glyoxal-, 3-deoxyglucosone/3-deoxygalactosone- and 3-deoxylactosone-derived dihydroxyimidazolines and/or hemiaminals were the most frequent derivatives observed. Proteins involved in nutrient delivery, defense response against pathogens and cellular proliferation/differentiation were highly subjected to intermediate and advanced glyco-oxidation modification. This may lead to a progressive decrease of the milk nutritional value, as it reduces the protein functional properties, diminishes the bioavailability of the essential amino acids, eventually affects food digestibility and determines a potential increase of specific allergens. These information are important points of interest to connect the extent of the Maillard reaction present in different commercial samples with the potential nutritional aspects mentioned above. These themes have to be fully evaluated in a next future for a complete estimation of the nutritional and toxicological properties of the dairy products deriving from severe heat processing.

Plasma protein changes in horse after prolonged physical exercise: a proteomic study.

Physical exercise induces various stress responses and metabolic adaptations that have not yet been completely elucidated. Novel biomarkers are needed in sport veterinary medicine to monitor training levels and to detect subclinical conditions that can develop into exercise-related diseases. In this study, protein modifications in horse plasma induced by prolonged, aerobic physical exercise were investigated by using a proteomic approach based on 2-DE and combined mass spectrometry procedures. Thirty-eight protein spots, associated with expression products of 13 genes, showed significant quantitative changes; spots identified as membrane Cu amine oxidase, α-1 antitrypsin, α-1 antitrypsin-related protein, caeruloplasmin, α-2 macroglobulin and complement factor C4 were augmented in relative abundance after the race, while haptoglobin β chain, apolipoprotein A-I, transthyretin, retinol binding protein 4, fibrinogen γ chain, complement factor B and albumin fragments were reduced. These results indicate that prolonged physical exercise affects plasma proteins involved in pathways related to inflammation, coagulation, immune modulation, oxidant/antioxidant activity and cellular and vascular damage, with consequent effects on whole horse metabolism.