Pascale Sarni-Manchado

A-type proanthocyanidins from pericarp of Litchi chinensis

Abstract A tannin extract was isolated from freeze-dried litchi pericarp (Litchi chinensis). HPLC analysis, after thioacidolysis reaction, revealed that this tannin fraction consisted of epicatechin units linked by both A- and B-type interflavonoid bonds. The calculated average degree of polymerization, considering the presence of A-type linkages, was 6.4. Characterization of oligomeric and polymeric procyanidins was performed using electrospray ionization mass spectrometry. Numerous oligomers containing one or more A-type interflavanoid linkages were detected. A regular repartition of the number of A-type linkages for each polymer length (e.g. from 2 to 7 for DP17 with mostly 4 A-type linkages) was observed. The average number of A-type linkages also increased with the DP, the predominant species containing one A-type linkage (denoted 1A) from DP2 to 5, 2A from DP6 to 10, 3A from DP11 to 15 and 4A from DP16 to 20. The highest DP was estimated at 22, with predominantly 5–6 A-type interflavanoid linkages.

Folding of a Salivary Intrinsically Disordered Protein upon Binding to Tannins

We used ion mobility spectrometry to explore conformational adaptability of intrinsically disordered proteins bound to their targets in complex mixtures. We investigated the interactions between a human salivary proline-rich protein IB5 and a model of wine and tea tannin: epigallocatechin gallate (EgCG). Collisional cross sections of naked IB5 and IB5 complexed with N = 1-15 tannins were recorded. The data demonstrate that IB5 undergoes an unfolded to folded structural transition upon binding with EgCG.

Influence of the Glycosylation of Human Salivary Proline-Rich Proteins on Their Interactions with Condensed Tannins

Binding of condensed tannins to salivary proteins is supposed to be involved in their astringency. First, complexes arising from the interaction of saliva from two individuals and tannins were studied. Then interaction mixture models containing purified saliva proteins were developed. The highest polymerized tannins predominantly precipitated together with the salivary proteins. Electrophoresis of proteins in combination with thiolysis analysis of tannins indicated proline-rich protein (PRP)-polyphenol complexes in precipitated fractions and also in the soluble ones with individual differences. Individual salivas exhibiting different protein patterns were discriminated with regard to their ability to interact with tannins. From binding studies with purified classes of salivary proteins, interactions were shown to depend on the nature of the protein, in particular on their glycosylation state. For low concentrations of tannins, glycosylated PRP-tannin interactions led to complexes that remained soluble, whereas those arising from nonglycosylated PRP-tannin interactions were precipitated. This finding could indicate that under physiological conditions, complexes involving glycosylated proteins maintain part of the lubrication of the oral cavity, whereas tannin trapping leads to a lower astringency perception.

Reactions of polyphenoloxidase generated caftaric acid o-quinone with malvidin 3-O-glucoside

Abstract Polyphenol oxidase (PPO-catalysed oxidation of solutions containing malvidin 3- O -glucoside and caftaric acid was studied by HPLC and mass spectrometry. The reaction of the anthocyanin with o -quinones and the nature of the products formed were compared as two pH values. The oxidized solution contained, in addition to the precursors, coloured and colourless compounds which eluted between caftaric acid and malvidin 3- O -glucoside. Liquid chromatography with ion spray mass spectrometry (LC-ISP-MS) indicated that they were adducts of malvidin 3- O -glucoside and caftaric acid, either in the flavylium or in the hemiacetal form.

Aggregation of the Salivary Proline-Rich Protein IB5 in the Presence of the Tannin EgCG

In the mouth, proline-rich proteins (PRP), which are major components of stimulated saliva, interact with tannins contained in food. We report in vitro interactions of the tannin epigallocatechin gallate (EgCG), with a basic salivary PRP, IB5, studied through electrospray ionization mass spectrometry (ESI-MS), small-angle X-ray scattering (SAXS), and dynamic light scattering (DLS). In dilute protein (IB5) solutions of low ionic strength (1 mM), the proteins repel each other, and the tannins bind to nonaggregated proteins. ESI-MS experiments determine the populations of nonaggregated proteins that have bound various numbers of tannin molecules. These populations match approximately the Poisson distribution for binding to n = 8 sites on the protein. MS/MS experiments confirm that complexes containing n = 1 to 8 EgCG molecules are dissociated with the same energy. Assuming that the 8 sites are equivalent, we calculate a binding isotherm, with a binding free energy Δμ = 7.26RT(a) (K(d) = 706 μM). In protein solutions that are more concentrated (0.21 mM) and at higher ionic strength (50 mM, pH 5.5), the tannins can bridge the proteins together. DLS experiments measure the number of proteins per aggregate. This number rises rapidly when the EgCG concentration exceeds a threshold (0.2 mM EgCG for 0.21 mM of IB5). SAXS experiments indicate that the aggregates have a core-corona structure. The core contains proteins that have bound at least 3 tannins and the corona has proteins with fewer bound tannins. These aggregates coexist with nonaggregated proteins. Increasing the tannin concentration beyond the threshold causes the transfer of proteins to the aggregates and a fast rise of the number of proteins per aggregate. A poisoned growth model explains this fast rise. Very large cationic aggregates, containing up to 10,000 proteins, are formed at tannin concentrations (2 mM) slightly above the aggregation threshold (0.2 mM).

Ability of a salivary intrinsically unstructured protein to bind different tannin targets revealed by mass spectrometry

AbstractAstringency is thought to result from the interaction between salivary proline-rich proteins (PRP) that belong to the intrinsically unstructured protein group (IUP), and tannins, which are phenolic compounds. IUPs have the ability to bind several and/or different targets. At the same time, tannins have different chemical features reported to contribute to the sensation of astringency. The ability of both electrospray ionization mass spectrometry and tandem mass spectrometry to investigate the noncovalent interaction occurring between a human salivary PRP, IB5, and a model tannin, epigallocatechin 3-O-gallate (EgCG), has been reported. Herein, we extend this method to study the effect of tannin chemical features on their interaction with IB5. We used five model tannins, epigallocatechin (EgC), epicatechin 3-O-gallate (ECG), epigallocatechin 3-O-gallate (EgCG), procyanidin dimer B2 and B2 3′-O-gallate, which cover the main tannin chemical features: presence of a gallate moiety (galloylation), the degree of polymerization, and the degree of B ring hydroxylation. We show the ability of IB5 to bind these tannins. We report differences in stoichiometries and in stability of the IB5•1 tannin complexes. These results demonstrate the main role of hydroxyl groups in these interactions and show the involvement of hydrogen bonds. Finally, these results are in line with sensory analysis, by Vidal et al. (J Sci Food Agric 83:564–573, 2003) pointing out that the chain length and the level of galloylation are the main factors affecting astringency perception. FigureCID MS/MS approach to monitor the stability of noncovalent complexes between a human salivary proline-rich protein and model tannins that cover the main chemical features of tannins

Characterization, stoichiometry, and stability of salivary protein–tannin complexes by ESI-MS and ESI-MS/MS

AbstractNumerous protein–polyphenol interactions occur in biological and food domains particularly involving proline-rich proteins, which are representative of the intrinsically unstructured protein group (IUP). Noncovalent protein–ligand complexes are readily detected by electrospray ionization mass spectrometry (ESI-MS), which also gives access to ligand binding stoichiometry. Surprisingly, the study of interactions between polyphenolic molecules and proteins is still an area where ESI-MS has poorly benefited, whereas it has been extensively applied to the detection of noncovalent complexes. Electrospray ionization mass spectrometry has been applied to the detection and the characterization of the complexes formed between tannins and a human salivary proline-rich protein (PRP), namely IB5. The study of the complex stability was achieved by low-energy collision-induced dissociation (CID) measurements, which are commonly implemented using triple quadrupole, hybrid quadrupole time-of-flight, or ion trap instruments. Complexes composed of IB5 bound to a model polyphenol EgCG have been detected by ESI-MS and further analyzed by MS/MS. Mild ESI interface conditions allowed us to observe intact noncovalent PRP–tannin complexes with stoichiometries ranging from 1:1 to 1:5. Thus, ESI-MS shows its efficiency for (1) the study of PRP–tannin interactions, (2) the determination of stoichiometry, and (3) the study of complex stability. We were able to establish unambiguously both their stoichiometries and their overall subunit architecture via tandem mass spectrometry and solution disruption experiments. Our results prove that IB5·EgCG complexes are maintained intact in the gas phase. FigureSchematic illustrating the interaction between IB5 and EgCG, leading to IB5·EgCG complexes that remain intact in the gas phase during ESI-MS analysis. This system provides a model of biological interest with regards to astringency

Photodissociation and Dissociative Photoionization Mass Spectrometry of Proteins and Noncovalent Protein–Ligand Complexes

Weight of evidence: Using synchrotron radiation, photo-fragmentation of an intrinsically disordered protein is probed and compared with classical tandem mass-spectrometry activation techniques. It provides excellent sequence coverage allowing the identification of the protein noncovalent binding sites.