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International audience; Condensed tannins undergo many modifications during winemaking and the aging of wine. These reactions take place between the various constituent units of the tannins as well with other types of molecules present in wines. This work aimed to highlight the potential reactions of tannins and aromas. After the monomeric standards of the reaction products were synthesized, the study of the reactivity of tannins (dimer B2, trimer C1, and grape seed tannins composed of 23% epicatechin-3-O-gallate) with aromas (3-sufanylhexan-1-ol, 4-methyl-4-sulfanylpentan-2-one, ethanethiol, and furfurylthiol) permitted identification of numerous reaction products. These tannin/aroma oxidation markers, detected after chemical depolymerization, allow for progress in the characterization of tannins in the oenological environment and the understanding of their reactivity toward the molecules present in the samples. A better characterization of tannins will make it possible to better measure the impact of these modifications on the organoleptic qualities of wines.

In order to obtain information on the oxidative behavior of red wines, oxygen consumption rates and electrochemical changes (cyclic voltammetry) were measured for nine red wines subject to three different accelerated ageing tests: chemical (with hydrogen peroxide), enzymatic (with laccase from Trametes versicolor), and temperature (at 60 °C). Oxidative behavior depended both on the wine sample and accelerated ageing test type. A good correlation was observed between electrochemical parameters of charges for reference/non-oxidized wines, in accordance with their antioxidant capacity, and the variation of charges after enzymatic and temperature tests, meaning that cyclic voltammetry could be used in order to predict these two oxidation tests and reflect the wine sensitivity towards respective oxidation targets. However, it was not possible to predict wine chemical oxidation test based on hydrogen peroxide from the electrochemical measurements.

B-type procyanidin dimers and (+)-catechin dimeric oxidation products were analyzed in grape seed extracts and red wines (UHPLC-Q-Orbitrap MS). The different dimers had different fragmentation patterns according to their interflavan linkage position. Oxidation dimeric compounds had a specific fragment ion at m/z 393, missing for B-Type dimers fragmentations. A fragment ion at m/z 291 occurred and was specific for oxidation dimeric compounds with a COC linkage. Higher level oxidation products had abundant specific fragments: m/z 425, 397 and 245. These fragmentations were useful to identify them in complex samples such as grape seed extracts and wines. Three grape varieties and three ripening stages were selected and the corresponding seed extracts were obtained. The analyses revealed an increasing trend for the oxidation markers during grape ripening. The analysis of Syrah wines (2018, 2014, 2010) showed a decreasing trend of these molecules during wine ageing which might be due to further oxidation.

(+)-Catechin—laccase oxidation dimeric standards were hemi-synthesized using laccase from Trametes versicolor in a water-ethanol solution at pH 3.6. Eight fractions corresponding to eight potential oxidation dimeric products were detected. The fractions profiles were compared with profiles obtained with two other oxidoreductases: polyphenoloxidase extracted from grapes and laccase from Botrytis cinerea. The profiles were very similar, although some minor differences suggested possible dissimilarities in the reactivity of these enzymes. Five fractions were then isolated and analyzed by 1D and 2D NMR spectroscopy. The addition of traces of cadmium nitrate in the samples solubilized in acetone-d6 led to fully resolved NMR signals of phenolic protons, allowing the unambiguous structural determination of six reaction products, one of the fractions containing two enantiomers. These products can further be used as oxidation markers to investigate their presence and evolution in wine during winemaking and wine ageing.

Red wine is a very complex medium in which condensed tannins undergo many modifications during winemaking and bottle ageing. These reactions have an impact on the organoleptic properties. This work aimed to highlight tannins evolution related to wine evolution by studying three vintages of Syrah wines. An accelerated oxidation was also undertaken in order to evaluate the ability of this oxidation to imitate natural evolution. After chemical depolymerization of the tannins, the monitoring of 6 types of markers at two oxidation levels was investigated. An evolution of the tannin oxidation state during ageing evidenced by the increase of the markers of the second oxidation level was observed. In the 2018 oxidized wine sample, the first oxidation level markers were similar to the 2014 vintage but the second oxidation level markers were higher than other vintages, indicating a more advanced state of tannin oxidation.

Wine oxidation and ageing involve many complex chemical pathways and reaction mechanisms. The purpose of this study is to set up new and reproducible accelerated red wine ageing tests and identify chemical oxidation or ageing molecular markers. Three accelerated and reproducible ageing tests were developed: a heat test (60 &deg, C), an enzymatic test (laccase test, a chemical test (hydrogen peroxide test). Depending on the test, oxygen consumption was significantly different. For a young wine (2018), the oxygen consumption rate moved from 2.40 ppm.h&minus, 1 for the heat test to 3.33 ppm.h&minus, 1 for the enzymatic test and 2.86 ppm.h&minus, 1 for the chemical test. Once applied to two other vintages (2010 and 2014) from the same winery, the tests revealed different comportments corresponding to wine natural evolution. High resolution UPLC-MS was performed on forced ageing samples and compared to naturally aged red wines. Specific oxidation or ageing ion markers were found with significant differences between tests, revealing the specificity of each test and different possible molecular pathways involved. The hydrogen peroxide test seems to be closer to natural oxidation with an important decrease in absorbance at 520 nm and similar molecular ion variations for [M+H]+ = 291, 331, 347, 493, 535, 581, 639 Da.

International audience; Rationale: The analysis of tannins is currently usually done by liquid chromatography after the chemical depolymerization of tannins. However, one of the main limitations of this method is the difficulty in specifically detecting each constitutive tannin monomer in a complex matrix, as numerous compounds co-elute with the monomers, thereby compromising the analysis.Methods: The depolymerization of tannins under acidic conditions and in the presence of thioglycolic acid releases the various constitutive monomers, either as terminal units or as extension units. The tannin subunits were then quantified using extracted-ion chromatogram (EIC) mass spectrometry, which required the determination of the ionization efficiency of the monomers. Thus, we used AgBF4-assisted coupling for the hemisynthesis of the noncommercial epicatechin gallate dimer.Results: The EIC showed that the derivatives of the extension units were twice as ionized as the terminal units. Unlike the UV chromatograms, this new EIC-based method is more specific and accurate because it is not impacted by the co-elution phenomenon. This result, when added to the linearity obtained on a large range of the calibration curves, allowed for quantification of tannin subunits from EIC in complex mixtures with less pretreatment of the samples.Conclusions: Due to its specificity and sensitivity, this EIC-based method represents a significant step toward improving the quantification of the tannin composition of samples. The results of this study should allow the oxidation markers to be quantified more accurately and taken into account, thereby providing a better indication of the actual tannin composition.

To improve the sensory quality of Moroccan red press wines, press wines were separately submitted to micro-oxygenation and three fining agents (gelatin, Polyvinylpolypyrrolidone (PVPP) based formulation and pea protein) treatments at the winery Chateau Roslane, Morocco. The results showed that each treatment has a distinct behavior in relation with interactions and precipitation monomeric fiavan-3-ol and fractions apparent and total condensed tannins. The fining agent corresponding to the vegetable protein has a greater affinity with the fraction of oligomeric condensed tannins with apparent aDP: 3.2. However, gelatin and PVPP based fining agent have an affinity with the catechin and epicatechin as well as with the fraction of polymeric tannins (apparent aDP: 6.6). For the structural parameters of proanthocyanidins, no significant differences were found out in global aDP and percentage of prodelphinidin between the different treatments. The global percentage of galloylation appeared slightly affected, while the global percentage of oxidized proanthocyanidins increased (9%) significantly (P < 0,05) for micro oxygenation treatment, unlike the fining agent has basic vegetable protein has decreased 36% the percentage of oxidation. For the Saliva Precipitation Index (SPI) and sensory analysis, the greatest reduction of SPI, bitterness and astringency was observed for both basic PVPP and vegetable protein fining agents.

Proanthocyanidins (PAs) and anthocyanins contents of four different red grape pomaces (GPs) from Morocco were analysed using chromatographic methods. The GP skin pigments characteristics were determined using spectroscopic methods while their antioxidant activities were determined using both spectroscopic methods as ABTS and DPPH radical cations, ferric reducing/antioxidant power (FRAP) and electrochemical technique: cyclic Voltammetry (CV). The PA of seeds extracts varied significantly among the types of GP and ranged from 56.1 +/- 0.3 mg g(-1) dry weight (DW) in Cot to 88.4 +/- 2.3 mg g(-1) DW in non-fermented Cabernet Sauvignon (CSNF). The skins of CSNF contained the highest levels of PAs (22.1 +/- 0.3 mg g(-1) DW). The PAs average degree of polymerization distribution ranged from 2 to 45 subunits for the skins and from 2 to 12 subunits for the seeds. The maximum anthocyanins amounts and antioxidant activities were measured in Cot skin extracts (32.8 mg g(-1) DW, DPPH: 0.23 mmol Trolox mg g(-1) DW; ABTS: 0.38 mmol Trolox mg g(-1) DW and CV: 1.73 x 10(-3)mA V) followed by Arinarnoa skin extracts which had the maximum corrected pigments (14.8 +/- 0.1) and color power (152.4 +/- 4.6 Unit color). The principal components analysis (PCA) of GP skins showed three different groups relevant to anthocyanins, PAs content, color and antioxidant properties.

Condensed tannins take an important part in the sensory quality of food and beverage. Sensory analyses are usually carried out with various tannin fractions isolated from food or beverage, and their interpretation are limited by the lack of knowledge in the fine and accurate molecular composition of the tannin fractions. Besides, the studies of the chemical reactivity conducted in model solutions with ‘simple’ flavanols allow a better understanding of their evolution pathways, but they cannot take into account their reactivity as polymers, specifically regarding oxidation. In particular, competition between intramolecular and intermolecular reactions may strongly impact on the tannin structures (size, branching and conformation) and consequently on their properties. An ultra-performance liquid chromatography-mass spectrometry electrospray ionization mass spectrometer analytical method was thus developed in order to identify oxidized tannins generated by autoxidation. Given the difficulties to separate and detect tannins with high DP, samples were depolymerized by chemical depolymerization prior to analysis. Since the linkages created by oxidation are not cleavable in the usual depolymerization conditions (contrarily to the original interflavanic linkages), specific oxidation residues are released from tannins structures after their autoxidation. Oxidation markers of both intermolecular and intramolecular mechanisms have been identified; these are mainly dimers and trimers, more or less oxidized, and some contain additional hydroxyl groups. Furthermore, the nature of the subunits (extension vs terminal) making up these dimers and trimers was clearly established. Copyright © 2012 John Wiley & Sons, Ltd.

Protein oxidation is thought to contribute to a number of inflammatory diseases, hence the development of sensitive and specific analytical techniques to detect oxidative PTMs (oxPTMs) in biological samples is highly desirable. Precursor ion scanning for fragment ions of oxidized amino acid residues was investigated as a label-free MS approach to mapping specific oxPTMs in a complex mixture of proteins. Using HOCl-oxidized lysozyme as a model system, it was found that the immonium ions of oxidized tyrosine and tryptophan formed in MS(2) analysis could not be used as diagnostic ions, owing to the occurrence of isobaric fragment ions from unmodified peptides. Using a double quadrupole linear ion trap mass spectrometer, precursor ion scanning was combined with detection of MS(3) fragment ions from the immonium ions and collisionally-activated decomposition peptide sequencing to achieve selectivity for the oxPTMs. For chlorotyrosine, the immonium ion at 170.1 m/z fragmented to yield diagnostic ions at 153.1, 134.1, and 125.1 m/z, and the hydroxytyrosine immonium ion at 152.1 m/z gave diagnostic ions at 135.1 and 107.1 m/z. Selective MS(3) fragment ions were also identified for 2-hydroxytryptophan and 5-hydroxytryptophan. The method was used successfully to map these oxPTMs in a mixture of nine proteins that had been treated with HOCl, thereby demonstrating its potential for application to complex biological samples.

In inflammatory diseases, release of oxidants leads to oxidative damage to proteins. The precise nature of oxidative damage to individual proteins depends on the oxidant involved. Chlorination and nitration are markers of modification by the myeloperoxidase-H2O2-Cl- system and nitric oxide-derived oxidants, respectively. Although these modifications can be detected by western blotting, currently no reliable method exists to identify the specific sites damage to individual proteins in complex mixtures such as clinical samples. We are developing novel LCMS2 and precursor ion scanning methods to address this. LC-MS2 allows separation of peptides and detection of mass changes in oxidized residues on fragmentation of the peptides. We have identified indicative fragment ions for chlorotyrosine, nitrotyrosine, hydroxytyrosine and hydroxytryptophan. A nano-LC/MS3 method involving the dissociation of immonium ions to give specific fragments for the oxidized residues has been developed to overcome the problem of false positives from ions isobaric to these immonium ions that exist in unmodified peptides. The approach has proved able to identify precise protein modifications in individual proteins and mixtures of proteins. An alternative methodology involves multiple reaction monitoring for precursors and fragment ions are specific to oxidized and chlorinated proteins, and this has been tested with human serum albumin. Our ultimate aim is to apply this methodology to the detection of oxidative post-translational modifications in clinical samples for disease diagnosis, monitoring the outcomes of therapy, and improved understanding of disease biochemistry.

Urinary proteomics is emerging as a powerful non-invasive tool for diagnosis and monitoring of variety of human diseases. We tested whether signatures of urinary polypeptides can contribute to the existing biomarkers for coronary artery disease (CAD). We examined a total of 359 urine samples from 88 patients with severe CAD and 282 controls. Spot urine was analyzed using capillary electrophoresis on-line coupled to ESI-TOF-MS enabling characterization of more than 1000 polypeptides per sample. In a first step a "training set" for biomarker definition was created. Multiple biomarker patterns clearly distinguished healthy controls from CAD patients, and we extracted 15 peptides that define a characteristic CAD signature panel. In a second step, the ability of the CAD-specific panel to predict the presence of CAD was evaluated in a blinded study using a "test set." The signature panel showed sensitivity of 98% (95% confidence interval, 88.7-99.6) and 83% specificity (95% confidence interval, 51.6-97.4). Furthermore the peptide pattern significantly changed toward the healthy signature correlating with the level of physical activity after therapeutic intervention. Our results show that urinary proteomics can identify CAD patients with high confidence and might also play a role in monitoring the effects of therapeutic interventions. The workflow is amenable to clinical routine testing suggesting that non-invasive proteomics analysis can become a valuable addition to other biomarkers used in cardiovascular risk assessment.

Model solutions of oxidized monomer/dimer mixtures of flavan-3-ol (EC/B2, EGC/B2, ECG/B2, EGCG/B2) were studied after chemical depolymerization by UPLC–MS. New oxidation markers of condensed tannins were identified. This analytical method was applied to a wine sample and most of these oxidized markers were detected. This method will permit following the oxidation state of tannins during the winemaking process and storage in order to improve the quality of the products.

The synthesis of a modified pentapeptide involving the palmitoylation of the hydroxyl group of a serine residue present at the N-terminal position is presented. An O–N-acyl shift was observed by LC/MS/MS, the two isobaric molecules exhibiting upon collisional activation dissociation (CAD) different fragmentation behaviours. The synthetic pathway was thereafter modified to control the palmitoylation site (O or N). The method was validated with another serine acylation (octanoylation). The evidenced mass spectrometric criteria could serve to decipher peptide post-translational modifications in proteomics.

In the context of the potential health benefits of food polyphenols, the bioavailability of tannins (i.e. proanthocyanidins) is a major issue, which is strongly influenced by the polydispersity and the degree of polymerisation of tannins. The average degree of polymerisation (DP) of tannins is usually determined using depolymerisation methods, which do not provide any information about their polymer distribution. Moreover, it is still a challenge to characterise tannin fractions of high polydispersity and/or containing polymers of high molecular weights, due to the limit of detection of direct mass spectrometry (MS) analysis methods. In the present work, the polydispersity of several tannin fractions is investigated by two complementary methods: a MALDI-MS method and a semi-preparative sub-fractionation. Using a combination of these methods we are able to gain insight into the DP distributions of the fractions consisting of tannins of medium and high DP. Moreover combining analyses can be useful to assess and compare the DP distributions of most tannin fractions.

Development of mass spectrometry techniques to detect protein oxidation, which contributes to signalling and inflammation, is important. Label-free approaches have the advantage of reduced sample manipulation, but are challenging in complex samples owing to undirected analysis of large data sets using statistical search engines. To identify oxidised proteins in biological samples, we previously developed a targeted approach involving precursor ion scanning for diagnostic MS3 ions from oxidised residues. Here, we tested this approach for other oxidations, and compared it with an alternative approach involving the use of extracted ion chromatograms (XICs) generated from high-resolution MSMS data using very narrow mass windows. This accurate mass XIC data methodology was effective at identifying nitrotyrosine, chlorotyrosine, and oxidative deamination of lysine, and for tyrosine oxidations highlighted more modified peptide species than precursor ion scanning or statistical database searches. Although some false positive peaks still occurred in the XICs, these could be identified by comparative assessment of the peak intensities. The method has the advantage that a number of different modifications can be analysed simultaneously in a single LC-MSMS run. This article is part of a Special Issue entitled: Posttranslational Protein modifications in biology and Medicine. Biological significance: The use of accurate mass extracted product ion chromatograms to detect oxidised peptides could improve the identification of oxidatively damaged proteins in inflammatory conditions. © 2013 Elsevier B.V.

Condensed tannins take an important part in the sensory quality of food and beverage. Sensory analyses are usually carried out with various tannin fractions isolated from food or beverage, and their interpretation are limited by the lack of knowledge in the fine and accurate molecular composition of the tannin fractions. Besides, the studies of the chemical reactivity conducted in model solutions with 'simple' flavanols allow a better understanding of their evolution pathways, but they cannot take into account their reactivity as polymers, specifically regarding oxidation. In particular, competition between intramolecular and intermolecular reactions may strongly impact on the tannin structures (size, branching and conformation) and consequently on their properties. An ultra-performance liquid chromatography-mass spectrometry electrospray ionization mass spectrometer analytical method was thus developed in order to identify oxidized tannins generated by autoxidation. Given the difficulties to separate and detect tannins with high DP, samples were depolymerized by chemical depolymerization prior to analysis. Since the linkages created by oxidation are not cleavable in the usual depolymerization conditions (contrarily to the original interflavanic linkages), specific oxidation residues are released from tannins structures after their autoxidation. Oxidation markers of both intermolecular and intramolecular mechanisms have been identified; these are mainly dimers and trimers, more or less oxidized, and some contain additional hydroxyl groups. Furthermore, the nature of the subunits (extension vs terminal) making up these dimers and trimers was clearly established.

The determination of the molecular mass distribution of tannins is still a challenge. To elucidate it, mass spectrometry is potentially interesting, but many previous studies have highlighted that the mass spectra of a tannin fraction do not always reflect the actual abundance of different chain lengths. To clarify the potentialities offered by the MS approach, a comprehensive study involving different tannin fractions analysed under different conditions was conducted with an electrospray ionization (ESI) source. This study allowed optimised ESI-MS conditions to be established for analysing tannins but also it outlines the limits of detection encountered. If the detection of high molecular weight tannins seems difficult or even impossible, the spectral distortions brought about by this limitation are not totally related to the sole average degree of polymerisation of the tannin fraction studied but greatly depend on its polymer distribution. However, ESI-MS used under optimised conditions is a suitable method to study tannin composition of vegetable extracts which contain degree of polymerisations below 26.

154MF Times Cited:4 Cited References Count:51; Efficient peptide sequencing relies on both high quality MS/MS data acquisition and exhaustive knowledge of gas-phase dissociation mechanisms. We report our contribution to the elaboration of more comprehensive fragmentation models required for efficient automated MS/MS spectra interpretation. Following a statistical approach, various peptides (296 sequences of variable compositions and lengths) were prepared and subjected to low-energy collision-induced dissociations (CID) in an electrospray hybrid instrument (ESI-Q-q-Tof type mass spectrometer) that has retained relatively limited attention so far. Besides, our studies were focused on low molecular weight singly charged peptides that often failed to be identified by sequencing algorithms. Only half of the studied compounds showed charge directed dissociations in accordance with the mobile proton model producing fragment ions directly related to the primary sequence. For the peptides that did not exhibit the expected fragment ion series, alternative dissociation behaviors issued from complex rearrangements were evidenced.

The behavior of C-terminal amidated and carboxylated peptides upon low-energy collision-induced dissociation (CID) was investigated. Two sets of 76 sequences of variable amino acid compositions and lengths were synthesized as model compounds. In most cases, C-terminal amidated peptides were found to produce, upon CID, an abundant loss of ammonia from the protonated molecules. To validate such MS/MS signatures, the studied peptides contained amino acids that can potentially release ammonia from their side chains, such as asparagine, glutamine, tryptophan, lysine and arginine. Arginine, and to a lesser extent lysine, was shown to induce a competitive fragmentation leading to the loss of ammonia from their side chains, thus interfering with the targeted backbone neutral release. However, when arginine or lysine was located at the C-terminal position mimicking a tryptic digest, losses of ammonia from the arginine side chain and from the peptide backbone were completely suppressed. Such results were discussed in the frame of peptidomic or proteomic studies in an attempt to reveal the presence of C-terminal amidated peptides or proteins.