Article pubs.acs.org/Langmuir
Rapid Screening and Identification of New Soluble Tannin−Salivary Protein Aggregates in Saliva by Mass Spectrometry (MALDI-TOF-TOF and FIA-ESI-MS) M. R. Perez-Gregorio, N. Mateus, and V. de Freitas* Departamento de Quimica e Bioquimica, Faculdade de Ciências da Universidade do Porto, Rua Campo Alegre 687, 4169-007 Porto, Portugal S Supporting Information *
ABSTRACT: Astringency is mainly attributed to the interaction between tannins and salivary proteins. Proline-rich proteins, histatins, and statherins are supposed to be the most reactive salivary proteins. This study aims to contribute to the knowledge of the tannin−protein binding process in saliva. It was identified for the first time in several soluble tannin−human salivary protein aggregates. A rapid mass spectrometry analytical method (MALDITOF and FIA-ESI-MS) was developed to identify new soluble tannin−human salivary protein aggregates. Three different tanninsprocyanidin B3 (B3), procyanidin B2 gallate (B2G), and pentagalloylglucoside (PGG)were tested to elucidate the tannin selectivity toward histatins, proline-rich proteins, and statherins in human saliva. A greater number of aggregates with a higher molecular weight was found when PGG was tested while no difference in the number and molecular mass range was observed in B3 or B2G salivary protein aggregates. This study confirms for the first time the bilateral selectivity of tannins and protein to yield soluble tannin−human salivary protein complexes. The results confirm that B3 and B2G are more selective than PGG. Furthermore, the families of proteins involved in the majority of B3−salivary protein soluble aggregates were primarly histatins, followed by basic proline-rich proteins and statherins. When B2G was tested, basic proline-rich proteins were involved in a greater number of aggregates, followed by histatines and statherins. Basic proline-rich proteins were also the family of proteins that formed a greater number of PGG−salivary protein aggregates followed by statherins and histatins. Acidic prolinerich proteins and glucosilated proline-rich proteins formed fewer soluble aggregates regardless of the tannin tested. The aggregation process was also found to be influenced by tannin and protein polarity. Indeed, the protein/tannin ratio of soluble aggregates increased with the tannin polarity. On the other hand, the only amphiphilic salivary proteins studied (histatins) formed a greater number of aggregates with the least polar tannin tested (B3).
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the astringency sensation of foods and beverages.17,18,54 Salivary proteins have been grouped into five structurally related major classes, namely, histatins, proline-rich protein PRPs (divided into basic (bPRPs), acidic (aPRPs), and glycosylated (gPRPs)), statherins, mucins, and cystatins.55 The study of astringency has been mainly focused on the interaction of tannins with salivary proline-rich proteins,18,20−22 but several reports have revealed that histatins and statherins could also be involved in astringency taste sensation in foods such as tea or wine.21,23,24 The interaction between tannins and proteins in solution occurs since tannins are highly reactive especially toward sulfhydryl and amino groups of proteins. The ability of tannins to bind different proteins varies considerably and depends on the environment (temperature, pH, ionic strength, solvent, salt concentration (osmotic pressure affects protein aggregation per se), etc.), protein size, conformation, type of protein and
INTRODUCTION Phenolic compounds are widespread in the plant kingdom as a result of plant secondary metabolism. They have been a strong focus of research because of their health effects in the prevention and/or treatment of several chronic diseases. Anticarcinogenic, antiatherogenic, antitrombotic, anti-inflamatory, antiulcer, antiallergenic, anticoagulant, antimicrobial, and vasodilatory effects and analgesic activities have been attributed to the ingestion of phenolic compounds in humans.1−11 On the other hand, several harmful effects have been reported for these compounds. In fact, tannins were often described in the past as antinutritional factors because they could bind and precipitate proteins and many other organic compounds.12−14,54−58 These xenobiotic attributions include the inhibition of digestive enzymes, the formation of relatively less digestible complexes with dietary proteins, depressed growth in rats, altered food consumption, and acute hepatotoxicity.15,16 As a result of these binding processes, tannins can also interact with salivary proteins, and this interaction is supposed to be at the origin of © 2014 American Chemical Society
Received: June 5, 2014 Published: June 26, 2014 8528
dx.doi.org/10.1021/la502184f | Langmuir 2014, 30, 8528−8537
Langmuir
Article
The main goal of this study was to perform a rapid and simple analytical method to determine and identify the main salivary protein−tannin (B3/B2G/PGG) aggregates to assay the binding selectivity of these three tannins by MALDI-TOF and to verify these results by FIA- ESI-MS analysis.
protein concentration, type and structure of phenolic compounds.25,28 The ability of tannins to bind proteins is also dependent on the molecular weight of the tannin and the number of sites capable of interacting with the protein.26 Tannins can interact with proteins, forming soluble and insoluble complexes.27 The pellet contains the insoluble complexes whereas the soluble aggregates remain in the supernatant. In the past, the tannin−protein binding has been reported to occur in a nonspecific manner, but several reports contributed to indicate that specific interaction also occurs.21 In general, it has been found that proteins which are readily precipitated by tannin are large, have a high proline content, and lack of secondary or tertiary structure, although some of them may possess a polyproline helix.26 Interestingly, proteins with a high affinity for tannin share a high proline content with several plant proteins, such as tannin-associated proteins from sorghum grains, but little is known about the interaction of tannins with these proline-rich proteins.29 The insoluble tannin−protein complexes are of particular interest for researchers, and different assays have been developed.27,29−31 But as mentioned, human salivary proteins also interact with tannins to form soluble complexes.32,33 The understanding of astringency has been mistakenly focused on the study of insoluble complexes since effects related to tannin−protein interactions may not require the precipitation of the complexes. Over the past several years, the ability to characterize complex mixtures has greatly advanced. This has been especially true for complex biological samples and is a major enabler for the “omics” revolution. New technologies are now changing the scope of the questions that can be addressed in biology and are extending the measurements out of the classic model. Several studies have been undertaken on the binding of tannins to salivary proteins by different physicochemical techniques based on turbidity34 and the sample colloidal state such as dynamic light scattering (DLS), nephelometry, SAX, or FTIR.35 Some information about aggregate structure was investigated through NMR assays.36,37 The use of NMR to perform analytical methods to elucidate the structure of tannin−protein complexes has been restricted to a limited number of compounds because of solubility issues, especially at high concentrations in an aqueous medium. Therefore, the development of new techniques to yield structural information on tannin−protein complexes becomes crucial. The determination of proteins by SDS-PAGE gel electrophoresis in mixtures containing aggregates was often a tool to elucidate the proteins involved in the process.33,38 Moreover, tannins could be determined after ultrafiltration processes. There are only a few studies in which mass spectrometry is used to verify the aggregation of the salivary proline-rich protein with tannins based on model solutions39−41,58 but none using whole saliva. Mass spectrometry has been applied to the detection of noncovalent complexes; however, it will be necessary to show the specificity of the interaction in the gas phase and to be cautious in the structural analogy between gas-phase and solution complexes. This issue constitutes a general debate in the mass spectrometry of noncovalent complexes. Many studies have demonstrated that MALDI is a powerfull technique applied to the analysis of proteins and polymers. However, due to the diversity of analytes present in whole saliva and their different interactions with tannins, there is not an universal approach in MALDI MS to characterize saliva− tannin aggregates.
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EXPERIMENTAL SECTION
Materials. All organic solvents used in this study are of analytical grade or mass spectrometry grade for TOF or Orbitrap analysis and were purchased from Panreac (Castellar del Vallés, Barcelona, Spain). Milli-Q water was obtained by an internal water purification system from Millipore (Millerica, MA, USA). Trifluoroacetic acid, HPLC grade, was purchased from Sigma-Aldrich (USA). MALDI matrixes assayed (2,5-dihydroxybenzoic acid (DHB), α-cyano-4-hydroxycinnamic acid (CHCA), and 2′,4′,-dihydroxyacetophenone (DHAP)) were obtained from Bruker Daltonik GmbH (Bremen, Germany). Procyanidin B3 Synthesis (B3). Procyanidin B3 was obtained by hemisynthesis between (+) taxifolin and (+) catechin.42 Taxifolin (100 mg, 0.986 mmol) and catechin (186 mg-, 1.58 mmol) were dissolved in ethanol (20 mL), and this solution was degassed under argon for 10 min. A sodium borohydrate solution previously degassed under argon was dropped under nonoxidized conditions (Ar), and the solution was left for 15 min under magnetic agitation. After that, degassed (Ar) distilled water (13.5 mL) was added, and the pH was adjusted to 4.5 using an acetic acid 5% v/v solution. Finally, procyanidin B3 was extracted with ethyl acetate and purified with a Toyopearl gel column according to previous experience.43 Procyanidin B2-Gallate Isolation from Grape Seeds (B2G). Condensed tannins were extracted from Vitis vinifera grape seed extract. This extract was fractionated through a TSK Toyopearl HW40(s) gel column (100 mm × 10 mm i.d., with 0.8 mL for 3 min in methanol as the eluent), yielding two fractions according to the method described in the literature.45 The first fraction contains mainly catechins, procyanidin dimers, and their galloyl derivatives, and the second fraction contains galloylated procyanidin dimers, procyanidin trimers and their galloyl derivatives, and procyanidin tetramers. Procyanidin B2 gallate was isolated from fraction I of the grape seed extract purified on a Toyopearl gel column and purified by semipreparative HPLC according to the experimental conditions described elsewhere.46 1,2,3,4,5,6-Penta-O-galloyl-β-D-glucopyranose Synthesis (PGG). PGG was synthesized from tannic acid according to Chen and Hagerman (2004).43 Tannic acid (5.0 g) was dissolved (“methanolyzed”) in 70% methanol in acetate buffer (0.1 M, pH 5.0) at 65 °C for 15 h. After this time, the pH of the mixture was immediately adjusted to 6.0 with NaOH. Methanol was then removed from the mixture by evaporation under reduced pressure at a temperature
