Protein evolution in white wine during winemaking

Background and Aims: Grape proteins are responsible for the appearance of haziness in white wines during storage after bottling. However, only a few studies have approached the analysis of the fate of must proteins throughout the alcoholic fermentation. This study aimed to systematically investigate the daily variations in protein type and content during the fermentation in order to understand its influence on hazing potential and to attain some basic information to improve the practical management of grape proteins involved in the hazing of white wines. Methods and Results: The evolution of total soluble protein and individual protein fractions was studied in samples taken before, during and after alcoholic fermentation of a white grape must. The results were then related to variations in protein instability as measured by the heat test. Both the quantity of soluble protein and the protein instability increased during fermentation and then decreased after 1-month storage of the wine. Protein composition did not vary during fermentation as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and anion exchange chromatography (AEC). However, variations in the relative proportions of the six protein fractions obtainable by AEC were noted in the different samples. The contribution of each AEC protein fraction to wine instability was determined by considering both the intrinsic instability and the relative quantity of each of the individual protein fractions in the wine. It was demonstrated that the grape thaumatin-like protein VVTL1, as identified by mass spectrometry, showed the largest increase during fermentation and accounted for almost 40% of the heat-induced haze of the final wine. Moreover, the decreased protein instability noted after one month storage of the wine could be attributed to the stabilizing effect of polysaccharides released by the yeast cells. Conclusions: The quantity and relative proportion of soluble proteins vary during and after the alcoholic fermentation, as does their heat instability in wine. Grape VVTL1, constituting a large proportion of the total proteins in wine, seems to play a major role in protein haze formation. The release of yeast polysaccharides is related to an increased heat stability of total wine protein, despite the increase in the relative proportion of their most unstable component VVTL1. Therefore, the hazing potential of a white wine seems to be affected by variations in the relative proportions of its macromolecular components occurring in the early stages of winemaking. Significance of the Study: This study addressed for the first time the issue of the protein changing during the fermentation of white wine. The results obtained here offer useful information to aid understanding of the contribution of individual proteins to white wine instability, which can be applied for the improvement of the winemaking process. Abbreviations AEC anion-exchange chromatography; KDS potassium dodecyl sulfate; MS mass spectrometry; MW molecular weight, PAS periodic acid-Schiff stain, PR-proteins pathogenesis-related proteins, RT retention time, SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis, TL thaumatin-like, VVTL1 Vitis vinifera thaumatin-like protei

High Abundance Proteins Depletion vs Low Abundance Proteins Enrichment: Comparison of Methods to Reduce the Plasma Proteome Complexity

BACKGROUND: To date, the complexity of the plasma proteome exceeds the analytical capacity of conventional approaches to isolate lower abundance proteins that may prove to be informative biomarkers. Only complex multistep separation strategies have been able to detect a substantial number of low abundance proteins (<100 ng/ml). The first step of these protocols is generally the depletion of high abundance proteins by the use of immunoaffinity columns or, alternatively, the enrichment of by the use of solid phase hexapeptides ligand libraries. METHODOLOGY/PRINCIPAL FINDINGS: Here we present a direct comparison of these two approaches. Following either approach, the plasma sample was further fractionated by SCX chromatography and analyzed by RP-LC-MS/MS with a Q-TOF mass spectrometer. The depletion of the 20 most abundant plasma proteins allowed the identification of about 25% more proteins than those detectable following low abundance proteins enrichment. The two datasets are partially overlapping and the identified proteins belong to the same order of magnitude in terms of plasma concentration. CONCLUSIONS/SIGNIFICANCE: Our results show that the two approaches give complementary results. However, the enrichment of low abundance proteins has the great advantage of obtaining much larger amount of material that can be used for further fractionations and analyses and emerges also as a cheaper and technically simpler approach. Collectively, these data indicate that the enrichment approach seems more suitable as the first stage of a complex multi-step fractionation protocol

Interaction between alpha-lactalbumin and lipids: conformational features and effects on protein aggregation

The topic of this PhD project concerns aspects of the general problem of the protein folding and unfolding, in line with the research conducted in the laboratory of Protein Chemistry at CRIBI, where the activities were mostly performed. The mechanism of acquisition of the three-dimensional structure of proteins (folding) is an important biological event. It is generally a multi-stage process involving the formation of intermediates, which are partly folded states having some structural features of the native protein, but not the final side chains interactions that allow the protein to exert its specific function. The failure in achieving the correct folding (misfolding) may cause protein aggregation. In fact, partly folded proteins can easily self-assembly in regular insoluble aggregates (amyloid fibrils), which are associated with serious diseases, the so called amyloidosis (Chiti & Dobson, 2006). In particular, my PhD research was focused on ?-lactalbumin (?-LA), a milk metalloprotein, widely used as a model to study protein folding. A new wave of interest in this protein appeared in the last decade after the discovery of a variant of the protein that, besides its physiological role in lactation, is able to induce apoptosis of tumor cells (Svensson et al., 1999). The cytotoxic activity resides in the formation of a complex with a milk fatty acid, oleic acid (OA), named HAMLET (Human Alpha-lactalbumin Made LEthal to Tumour cells) (Svensson et al., 2000). The cell events caused by HAMLET were extensively studied (Kohler et al., 2001; Duringer et al., 2003). On the contrary, the mechanism and the nature of the interaction between ?-LA and OA and the physico-chemical properties of the complex are not completely understood yet. The preparation of the complex is a controversial aspect. Indeed, despite the effective association of OA with the protein in solution (Polverino de Laureto et al., 2002), the resulting ?-LA/OA complex was believed to possess lower antitumor activity than HAMLET, obtained using a chromatographic procedure (Svensson et al., 2000). Other debated matters focus on the protein/fatty acid stoichiometry and the monomeric/oligomeric state of ?-LA in the complex. Moreover, a systematic investigation of the effect of the protein on the phase behavior of OA is still lacking. The aim of this PhD Thesis was the investigation of the OA binding propensity and cytotoxic activity of three proteolytic derivatives of bovine ?-LA, obtained by limited proteolysis with pepsin at acidic pH. The use of proteolytic dissection of a protein has been widely employed to study the folding of several proteins (Wetlaufer, 1981; Gegg et al., 1997; Llinás & Marqusee, 1998). The characterization of protein fragments containing specific structural elements of the intact protein (?-helix, ?-sheet) and able of autonomous folding was successfully used to unravel folding features of ?-LA (Polverino de Laureto et al., 1999; 2001). Indeed, fragments corresponding to structural domains in multidomain proteins were shown in some cases to acquire in solution a native-like conformation (Fontana et al., 2004). The ?-LA fragments investigated are: species 1–40/53–123, lacking the ?-subdomain of the native protein; 1–40/104–123, given by the N-terminal fragment 1–40 covalently linked by two disulfide bridges to the C-terminal fragment 104–123 and containing three of the four ?-LA helices; 53–103, containing the C-helix and the calcium binding loop of intact ?-LA (Polverino de Laureto et al., 1999; 2001). The conformational differences between the three fragments were used as the rationale for a study of their efficacy to bind OA, thus giving an insight into the mechanism of this binding. This study has also some physiological relevance: pepsin is an enzyme of the stomach and therefore these species might be generated in vivo, under the same conditions of low pH in which the complex HAMLET was hypothesized to form, thus contributing to the apoptotic activity of the complex formed by the intact protein. The complexes between the three fragments and OA were prepared first by the chromatographic procedure described for HAMLET, then by directly mixing in solution the two components. The conformational properties of the complexes were characterized by circular dichroism, showing that the complexes prepared by both procedures display similar conformations and acquire ?-helix. The effect of calcium on the conformation of the complexes was then investigated by circular dichroism. Fluorescence spectroscopy was used to study the involvement of Trp residues in the interaction with OA. Moreover, the ability of the complexes to induce apoptosis-like cell death was evaluated, in line with the cytotoxic activity displayed by HAMLET. In order to verify the physical state of OA involved in the interaction with ?-LA, the OA aggregation behavior was investigated using different techniques, such as transmission electron microscopy (TEM), titration with a fluorescent dye and turbidimetric analyses, and an effect of solubilization of OA was observed. A manuscript with these results will be soon submitted to an international journal and is herewith attached. In the second part of the PhD, the research was focused on the aggregation propensity of ?-LA and its three proteolytic fragments. ?-LA is able to form amyloid fibrils in vitro, even if not disease-related. ?-LA fibrils are formed when the structure of the protein is partially destabilized, e.g. at low pH, upon reduction of three disulphide bridges at neutral pH (Goers et al., 2002), or after proteolytic cleavage (Polverino de Laureto et al., 2005). Lipids may act as an effective catalyst of fibrillogenesis, providing a generic environment where protein molecules adopt conformation and orientation promoting their assembly into fibrillar structures (Thirumalai et al., 2003; Stefani, 2004; Sparr et al., 2004; Zhao et al., 2004). In particular, since fatty acid–protein interactions are known to modulate the process of fibrillation (Kim & Takahashi 2006), aggregation processes of ?-LA and its fragments were investigated to elucidate whether and how OA might affect fibrils formation. Firstly, aggregation was followed at pH 2.0, as a comparison with the known fibrillogenic behavior of intact ?-LA. Secondly, the aggregation was investigated at pH 7.4, since in this physiological condition the conformational changes induced by OA on the fragments structure were studied and their cytotoxic activity was analyzed, in the first part of the Thesis. The formation of fibrils was followed by thioflavin T (ThT) fluorescence assay, circular dichroism and TEM. All three fragments were able to produce amyloid fibrils at pH 2.0, similarly to ?-LA. In the used range of protein concentration and protein/lipid ratio, OA seems to accelerate the rate of fibrillation for ?-LA and the three fragments at pH 2.0. At pH 7.4, ?-LA is not able to form fibrils both in the absence and in the presence of OA. Also the three fragments did not form amyloid fibrils at neutral pH, while in the presence of OA they underwent a conformational change to ?-sheet structure, were able to bind ThT and to form aggregates with the typical amyloid morphology. During the PhD course, I also collaborated to an ongoing project of the laboratory at CRIBI, focused on the characterization of oligomeric species on the aggregation pathway of human lysozyme, which belongs to the so called “lysozyme/lactalbumin superfamily”. Soluble oligomers of lysozyme were produced at low pH and high temperature, and then analyzed by a range of techniques including binding to fluorescent probes, Fourier-transform infrared (FTIR) spectroscopy and limited proteolysis. Oligomers have solvent-exposed hydrophobic patches, and FTIR spectra are indicative of highly misfolded species. Moreover, the oligomeric lysozyme aggregates were found to be more susceptible to proteolysis than both the monomeric protein and the mature fibrils, indicating their lack of organized structure. This study showed that the soluble lysozyme oligomers are structurally flexible species present at low concentration during the initial phases of aggregation. The results of this study were accepted for publication in an international journal and the ‘in press’ manuscript is attached to the Thesis. During the third year of PhD, I spent a six months period at the University of Cambridge (UK), in the Cambridge Centre for Proteomics, under the supervision of Dr. Kathryn Lilley. The aim of this period was to learn proteomic methodologies for large scale identification of proteins, improving my background in mass spectrometry. I was involved in an ongoing project of the laboratory, focused on a parallel affinity purification method coupled to mass spectrometry for identifying proteins and their binding partners in Drosophila melanogaster embryos (Veraksa et al., 2005). Triple tagged proteins were generated (Spradling et al., 1999) and isolated from a variety of tissues in embryos. Affinity purification using two tags in parallel allowed the isolation of intact native protein complexes. The high sensitivity and high mass accuracy of the hybrid LTQ-Orbitrap instrument ensured maximal coverage of low abundance complex components, generating high confident data with low false discovery rates. The software ProteinCenter™ (Proxeon) was utilized for the visualization and the statistical comparison of the datasets. The data were compared with datasets published in public databases, which validate the data and increased the certainty of the detected interaction sets. Also novel protein interactions not previously reported were mapped. These high confidence in vivo protein datasets add high confidence data to the currently incomplete D. melanogaster proteome and interactome. Here, the results of five different size proteins from different cellular localizations are reported in detail to show the workflow and the efficiency of the methodology. Summing up, this PhD Thesis is composed of a major part dealing with the characterization of the interaction of ?-LA and oleic acid and the effects on the protein aggregation, and a minor part dealing with the mass spectrometry analysis of Drosophila protein complexes, besides the publication on the oligomeric species in the aggregation pathway of lysozyme.L’argomento di questa Tesi di Dottorato riguarda in generale aspetti del problema del folding e unfolding proteico, in linea con la tematica di ricerca condotta nel laboratorio di Chimica delle Proteine al CRIBI, dove le attività sono state per la maggior parte svolte. Il meccanismo di acquisizione della struttura tridimensionale di una proteina (folding) è un evento biologico importante. In generale, è un processo multi-stadio che coinvolge la formazione di intermedi, che sono stati parzialmente strutturati contenenti alcune caratteristiche strutturali della proteina nativa, ma non le interazioni finali tra le catene laterali che permettono alla proteina di esercitare la sua funzione specifica. Il fallimento nell’acquisizione del corretto folding (misfolding) può causare aggregazione proteica. Infatti, proteine parzialmente strutturate possono facilmente auto-assemblarsi in aggregati regolari insolubili (fibrille amiloidi), associati a gravi malattie, le cosiddette amiloidosi (Chiti & Dobson, 2006). In particolare, il mio progetto di Dottorato è focalizzato sull’?-lattalbumina (?-LA), una metallo-proteina del latte, ampiamente utilizzata come modello di studio del folding proteico. Nell’ultimo decennio, questa proteina ha suscitato un nuovo interesse per la scoperta di una variante che, oltre al ruolo fisiologico nella lattazione, è in grado di indurre apoptosi nelle cellule tumorali (Svensson et al., 1999). L’attività citotossica risiede nella formazione di un complesso con un acido grasso del latte, l’acido oleico (OA), denominato HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells) (Svensson et al., 2000). Gli eventi cellulari causati da HAMLET sono stati estesamente studiati (Kohler et al., 2001; Duringer et al., 2003). Al contrario, il meccanismo e la natura dell’interazione tra ?-LA e OA e le proprietà fisico-chimiche del complesso non sono ancora completamente chiariti. Infatti, nonostante l’effettiva associazione dell’OA con la proteina in soluzione (Polverino de Laureto et al., 2002), si ritiene che il complesso così ottenuto possieda attività antitumorale minore dell’HAMLET, preparato secondo una procedura cromatografica (Svensson et al., 2000). Altre questioni dibattute riguardano la stechiometria proteina/acido grasso e lo stato monomerico/oligomerico dell’?-LA nel complesso. Inoltre, manca un’indagine sistematica dell’effetto della proteina sul comportamento di fase dell’OA. Lo scopo di questa Tesi di Dottorato è lo studio della capacità di legare OA ed esprimere attività citotossica di tre derivati proteolitici di ?-LA bovina, ottenuti per proteolisi limitata con pepsina a pH acido. L’uso di un approccio di proteolytic dissection di una proteina è stato largamente impiegato per studiare il folding di molte proteine (Wetlaufer, 1981; Gegg et al., 1997; Llinás & Marqusee, 1998). La caratterizzazione di frammenti proteici, contenenti elementi strutturali specifici della proteina intera (?-elica, ?-sheet) e in grado di assumere struttura in modo autonomo, è stata usata con successo per chiarire caratteristiche strutturali di ?-LA (Polverino de Laureto et al., 1999; 2001). Infatti, frammenti corrispondenti a domini strutturali in proteine multi-dominio hanno mostrato in alcuni casi la capacità di acquisire in soluzione una conformazione simile a quella nativa (Fontana et al., 2004). I frammenti di ?-LA studiati sono: la specie 1–40/53–123, priva di parte del dominio ? della?proteina nativa; 1–40/104–123, formato dal frammento N-terminale 1-40 legato covalentemente al frammento C-terminale 104-123 mediante due ponti disolfuro e contenente tre delle quattro ?-eliche di ?-LA; 53-103, contenente l’elica C e il sito di legame al calcio (Polverino de Laureto et al., 1999; 2001). Le differenze conformazionali dei tre frammenti sono state utilizzate come razionale per studiare la loro efficacia di legame all’OA, e per approfondire quindi il meccanismo di questo legame. Questo studio ha anche rilevanza fisiologica: la pepsina è un enzima dello stomaco e quindi queste specie potrebbero essere generate in vivo, nelle stesse condizioni di pH acido in cui si è ipotizzata la formazione del complesso HAMLET, contribuendo così all’attività apoptotica del complesso formato dalla proteina intera. I complessi dei tre frammenti con OA sono stati preparati prima seguendo la procedura cromatografica descritta per l’HAMLET, poi per diretto miscelamento in soluzione dei due componenti. Le proprietà conformazionali dei complessi sono state caratterizzate mediante dicroismo circolare, mostrando che i complessi preparati attraverso entrambe le procedure presentano conformazioni simili e acquisizione di ?-elica. Inoltre, è stato valutato l’effetto del calcio sulla conformazione dei complessi mediante dicroismo circolare. La spettroscopia di fluorescenza è stata utilizzata per analizzare il coinvolgimento dei residui di Trp nell’interazione con OA. Inoltre, è stata studiata la capacità dei complessi di indurre morte cellulare per apoptosi, in linea con l’attività citotossica mostrata dall’HAMLET. Per analizzare lo stato fisico dell’OA coinvolto nell’interazione con l’?-LA, il comportamento di aggregazione di OA è stato studiato con diverse tecniche, quali microscopia elettronica a trasmissione (TEM), titolazione con un colorante fluorescente e analisi turbidimetriche, ed è stato osservato un effetto di solubilizzazione dell’OA. Questi risultati hanno permesso di preparare un articolo che sarà presto spedito ad una rivista internazionale e che è allegato alla Tesi. Nella seconda parte del Dottorato, la ricerca è stata focalizzata sulla tendenza di ?-LA e dei suoi tre frammenti proteolitici ad aggregare. ?-LA è in grado di formare fibrille amiloidi in vitro, sebbene non sia associata a patologie. Fibrille di ?-LA si producono quando la struttura della proteina è parzialmente destabilizzata, ad esempio a pH acido, per riduzione di tre ponti disolfuro a pH neutro (Goers et al., 2002), o per taglio proteolitico (Polverino de Laureto et al., 2005). I lipidi possono agire come efficaci catalizzatori della fibrillogenesi, creando un ambiente in cui le molecole proteiche adottano una conformazione e un’orientazione che promuove il loro assemblaggio in strutture fibrillari (Thirumalai et al., 2003; Stefani, 2004; Sparr et al., 2004; Zhao et al., 2004). In particolare, poiché è noto che le interazioni proteina-acido grasso modulano il processo di fibrillogenesi (Kim & Takahashi 2006), i processi di aggregazione di ?-LA e dei suoi frammenti sono stati studiati per capire se e come l’OA possa influenzare la formazione di fibrille. In primo luogo, l’aggregazione è stata seguita a pH 2.0, in parallelo con il già noto comportamento fibrillogenico di ?-LA intera. In secondo luogo, l’aggregazione è stata studiata a pH 7.4, poiché in questa condizione fisiologica nella prima parte della Tesi sono stati studiati i cambiamenti conformazionali indotti dall’OA sulla struttura dei frammenti e la loro attività citotossica. La formazione di fibrille è stata seguita mediante saggi di fluorescenza con la tioflavina T (ThT), dicroismo circolare e TEM. Tutti e tre i frammenti sono in grado di produrre fibrille amiloidi a pH 2.0, analogamente all’?-LA. Ai valori di concentrazione proteica e rapporto proteina/lipide utilizzati, OA sembra accelerare la velocità di formazione di fibrille di ?-LA e dei frammenti a pH 2.0. A pH 7.4, ?-LA non forma fibrille amiloidi sia in assenza sia in presenza di OA. Anche i tre frammenti non sono stati in grado di formare fibrille a pH neutro, mentre in presenza di OA hanno mostrato un cambiamento conformazionale verso una struttura ?-sheet, hanno legato ThT e formato aggregati con la tipica morfologia amiloide. Durante il Dottorato, ho inoltre collaborato ad un progetto in corso nel laboratorio al CRIBI, relativo alla caratterizzazione di specie oligomeriche nel processo di aggregazione del lisozima umano, che appartiene alla cosiddetta “superfamiglia lisozima/lattalbumina”. Oligomeri solubili di lisozima sono stati prodotti a pH acido e alta temperatura, e quindi analizzati con varie tecniche, quali legame a molecole fluorescenti, spettroscopia infrarosso in trasformata di Fourier (FTIR) e proteolisi limitata. Gli oligomeri presentano superfici idrofobiche esposte al solvente, e gli spettri FTIR sono indicativi di specie altamente destrutturate. Inoltre, gli aggregati oligomerici di lisozima si sono rivelati più suscettibili alla proteolisi rispetto sia alla proteina monomerica sia alle fibrille mature, indicando la mancanza di una struttura organizzata. Questo studio ha dimostrato che gli oligomeri solubili di lisozima sono specie strutturalmente flessibili presenti a bassa concentrazione durante le fasi iniziali dell’aggregazione. I risultati di questo studio sono stati accettati per la pubblicazione in una rivista internazionale e il manoscritto in press è allegato alla Tesi. Durante il terzo anno di Dottorato, ho trascorso un periodo di sei mesi all’Università di Cambridge (UK), presso il Cambridge Centre for Proteomics, sotto la supervisione della Dr.ssa Kathryn Lilley. L’obiettivo di questo periodo è stato di apprendere metodologie di proteomica per l’identificazione di proteine su larga scala, ottimizzando le mie conoscenze di spettrometria di massa. Ho collaborato ad un progetto in corso nel laboratorio, incentrato su un metodo di purificazione di affinità in parallelo accoppiata a spettrometria di massa per identificare complessi proteici in embrioni di Drosophila melanogaster (Veraksa et al., 2005). Proteine con tre tags sono state generate (Spradling et al., 1999) e isolate da vari tessuti embrionali. La purificazione di affinità usando due tags in parallelo ha permesso di isolare complessi proteici nativi intatti. L’alta sensibilità e l’alta accuratezza di massa dello strumento ibrido LTQ-Orbitrap ha assicurato la massima copertura di componenti di complessi poco abbondanti, generando dati ad alta confidenza con basse false discovery rates. È stato utilizzato il software ProteinCenter™ (Proxeon) per la visualizzazione e l’analisi statistica dei set di dati. I risultati sono stati confrontati con dati presenti in database pubblici, confermandone la validità e aumentando l’autenticità delle interazioni individuate. Sono state inoltre mappate nuove interazioni proteiche non riportate in precedenza. Questi set di dati in vivo aggiungono alta confidenza al proteoma e ‘interattoma’ di D. melanogaster attualmente incompleto. In questa Tesi sono riportati in dettaglio i risultati di cinque proteine di diverse dimensioni e localizzazione cellulare, per presentare la procedura e l’efficienza della metodologia. In sintesi, questa Tesi di Dottorato è composta da una parte principale che riguarda la caratterizzazione dell’interazione tra ?-LA e acido oleico e gli effetti sull’aggregazione proteica, e una parte minore relativa all’analisi di spettrometria di massa di complessi proteici in Drosophila, oltre alla pubblicazione sulle specie oligomeriche studiate nel processo di aggregazione del lisozima

Characterization of chitinase isoforms from grape juice

Grape chitinases are recognized as being mainly responsible for protein haze formation in white wines. Vitis vinifera L. cv. Manzoni Bianco grape juice proteins were fractionated using anion exchange and hydrophobic interaction chromatographies. According to SDSPAGE and zymography, six protein bands with chitinolytic activity were subjected to mass spectrometry (MALDI-TOF/TOF MS), which assigned all the bands to Vitis vinifera class IV chitinases. These grape chitinase isoforms showing different electrophoretic and chromatographic behaviours are likely to be also distinct in their functionality in wine. This could be relevant to understand the involvement of single chitinase components in wine hazing and to develop specific winemaking techniques for their removal from wine

Caratterizzazione di isoforme di chitinasi da succo d\u2019uva

Le chitinasi dell\u2019uva sono considerate tra le principali responsabili della instabilit\ue0 proteica nei vini bianchi. In questo lavoro le proteine del mosto (cv. Manzoni bianco) sono state frazionate utilizzando una cromatografia a scambio anionico seguita da una cromatografia a interazione idrofobica. In base all\u2019analisi elettroforetica (SDS-PAGE) e alla rilevazione dell\u2019attivit\ue0 enzimatica su gel sono state identificate sei diverse bande proteiche con attivit\ue0 chitinolitica. Queste bande sono state sottoposte a spettrometria di massa (MALDI-TOF/TOF MS), che ha identificato tutte le frazioni come Chitinasi di classe IV di Vitis vinifera. Tali isoforme, che mostrano differenti caratteristiche elettroforetiche e cromatografiche, hanno probabilmente un diverso comportamento nel determinare gli intorbidamenti del vino. Quindi il loro studio \ue8 importante per meglio capire il coinvolgimento di ogni singola componente nella formazione di torbidit\ue0 e per sviluppare tecniche enologiche specifiche mirate alla prevenzione della casse proteica dei vini bianchi

Analysis of Commercial Wines by LC-MS/MS Reveals the Presence of Residual Milk and Egg White Allergens

Fining agents of animal origin are commonly used in winemaking process to clarify and stabilize wines and to optimize their organoleptic properties thanks to the removal of phenolic compounds that cause bitterness and astringency. Considering the potential allergenicity of proteins used as fining agents, the need of label declaration for wines treated with such compounds is compelling. However, the difficulties in detecting proteins in a wine matrix by immunological assays have given rise to a search for alternative methods that can overcome the limitations of classical approaches. Mass spectrometry (MS) has recently emerged as a powerful and sensitive technique to detect residual fining proteins in wines. In this study we show that a simple and straightforward mass spectrometric approach can be used to reliably detect egg and milk allergens in commercial bottled wines. We tested 25 different wines by liquid chromatography coupled with tandem MS (LC-MS/MS) and found proteins of animal origin in 8 samples, so demonstrating that the proposed method allows to monitor the presence of potential allergenic proteins in fined commercial wine

Characterization of Oligomeric Species on the Aggregation Pathway of Human Lysozyme

The aggregation process of wild-type human lysozyme at pH 3.0 and 60 °C has been analyzed by characterizing a series of distinct species formed on the aggregation pathway, specifically the amyloidogenic monomeric precursor protein, the oligomeric soluble prefibrillar aggregates, and the mature fibrils. Particular attention has been focused on the analysis of the structural properties of the oligomeric species, since recent studies have shown that the oligomers formed by lysozyme prior to the appearance of mature amyloid fibrils are toxic to cells. Here, soluble oligomers of human lysozyme have been analyzed by a range of techniques including binding to fluorescent probes such as thioflavin T and 1-anilino-naphthalene-8-sulfonate, Fourier transform infrared spectroscopy, and controlled proteolysis. Oligomers were isolated after 5 days of incubation of the protein and appear as spherical particles with a diameter of 8–17 nm when observed by transmission electron microscopy. Unlike the monomeric protein, oligomers have solvent-exposed hydrophobic patches able to bind the fluorescent probe 1-anilino-naphthalene-8-sulfonate. Fourier transform infrared spectroscopy spectra of oligomers are indicative of misfolded species when compared to monomeric lysozyme, with a prevalence of random structure but with significant elements of the β-sheet structure that is characteristic of the mature fibrils. Moreover, the oligomeric lysozyme aggregates were found to be more susceptible to proteolysis with pepsin than both the monomeric protein and the mature fibrils, indicating further their less organized structure. In summary, this study shows that the soluble lysozyme oligomers are locally unfolded species that are present at low concentration during the initial phases of aggregation. The nonnative conformational features of the lysozyme molecules of which they are composed are likely to be the factors that confer on them the ability to interact inappropriately with a variety of cellular components including membranes

Mass spectrometry detection of egg proteins in red wines treated with egg white

The need of label declaration for egg proteins is temporarily suspended when they are used as a processing aid in winemaking, because of a lack of scientific data concerning their actual permanence as residual proteins in fined wines. The possibility to detect residual egg proteins in red wines treated with a commercial egg white preparation was studied. By using an immunochemical method residual egg proteins were detected in the experimental red wines only for doses of fining agent of 50 g/hL or higher, whereas no residual proteins were detected by this system in a commercial red wine. A simple method based on the recovery and identification of the wine fining proteins by liquid chromatography coupled with tandem MS (LC-MS/MS) in a gel-free approach was developed. This allowed the detection of egg proteins in red wines fined down to 5 g/hL of commercial egg white preparation and also in the commercial red wine. These results indicate that the analytical approach here suggested is superior to the immunochemical methods in detecting egg proteins in wines. Therefore hypersensitivity reactions after consumption of wines treated with egg proteins can be a real risk for eggallergic people