Article pubs.acs.org/JAFC
New Procyanidin B3−Human Salivary Protein Complexes by Mass Spectrometry. Effect of Salivary Protein Profile, Tannin Concentration, and Time Stability Maria Rosa Perez-Gregorio, Nuno Mateus, and Victor 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: Several factors could influence the tannin−protein interaction such as the human salivary protein profile, the tannin tested, and the tannin/protein ratio. The goal of this study aims to study the effect of different salivas (A, B, and C) and different tannin concentrations (0.5 and 1 mg/mL) on the interaction process as well as the complex’s stability over time. This study is focused on the identification of new procyanidin B3−human salivary protein complexes. Thus, 48 major B3−human salivary protein aggregates were identified regardless of the saliva and tannin concentration tested. A higher number of aggregates was found at lower tannin concentration. Moreover, the number of protein moieties involved in the aggregation process was higher when the tannin concentration was also higher. The selectivity of the different groups of proteins to bind tannin was also confirmed. It was also verified that the B3−human salivary protein complexes formed evolved over time. KEYWORDS: procyanidin B3, salivary proteins, tannin−protein binding, astringency
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INTRODUCTION The main human salivary proteins are commonly grouped into six structurally related major classes, namely, histatins; prolinerich proteins (PRPs), which are divided into basic (bPRPs), acidic (aPRPs), and glycosylated (gPRPs) PRPs; statherin; and cystatins.1 In the mouth, salivary proteins interact with food tannins, and this is recognized as the most established mechanism of the astringency process. It is known that tannin−protein aggregation involves hydrophobic interactions and hydrogen bonding.2 These interactions depend on several factors such as the type and concentration of tannins and proteins,3,4 pH, and temperature. 5 The tannin−protein aggregation process is also affected by the protein/tannin ratio,6 and it has been proposed that the effectiveness of the interaction occurs at an optimum protein/tannin ratio. In fact, Hagerman and Robbins7 verified that tannin/protein aggregation follows a hyperbolic behavior. The farther is the tannin/ protein ratio from the optimum, the lower is the quantity of insoluble precipitates. This could be explained as a protein has a fixed number of sites to bind tannins and a tannin is thought to have two (or more) ends that could bind to protein. Thus, when the total concentration of tannin ends is equal to the number of protein binding sites, this results in a large network, corresponding to large colloidal particles and maximum precipitation. On the other hand, if there is a large excess of protein, each tannin molecule is able to bind two or more proteins.4 Furthermore, Canon et al.8 verified that in very dilute protein/tannin solutions of low ionic strength the proteins are repelled from each other. In these conditions the tannins bind to individual proteins but do not cause any aggregation. Several techniques have been used to study these interactions: NMR, circular dichroism, mass spectrometry, UV−vis and fluorescence spectroscopy, microcalorimetry, and flow nephelometric analysis.9 © 2014 American Chemical Society
A light scattering technique has been used as a direct measurement method to determine the size of aggregates of protein−tannin over a wide range of conditions before precipitation occurs.8 A great number of the works published concerning the astringency process were focused on studies on the insoluble protein−tannin aggregates. However, soluble tannin−protein aggregates could be very relevant. Thus, determining the factors that govern the formation of the soluble tannin−protein aggregates, namely, the influence of food polyphenols and salivary protein structures, concentration, molar ratio, kinetics of formation, and aggregate stability, became crucial to understand the astringency process. Peleg et al.9 evaluated the bitterness and astringency of flavan-3-ol monomers, dimers, and trimers, and they confirmed the B3 astringency through a sensorial project. In addition, Kallithraka et al.10 examined the proteins in saliva samples taken before and immediately after tasting of astringency solutions, and they found a new peak tentatively identified as soluble tannin−soluble protein complexes presumably related with the astringency sensation. Hence, the aim of this work was to determine the interaction of B3 with three different salivas with different salivary protein profiles at two different tannin concentrations. Moreover, the time stability of B3−salivary protein aggregates was also studied for one saliva and at one tannin fixed concentration.
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MATERIALS AND METHODS
Materials. All organic solvents used in this study are of analytical grade or mass spectrometry grade for TOF or ESI-MS analysis and were purchased from Panreac (Castellar del Vallés, Barcelona, Spain). Milli-Q water was obtained by an internal water purification system from Received: Revised: Accepted: Published: 10038
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Millipore (Billerica, MA, USA). Trifluoroacetic acid, HPLC grade, was purchased from Sigma-Aldrich (St. Louis, MO, USA). MALDI matrix (sinapinic acid) was obtained from BrukerDaltonik GmbH (Bremen, Germany) (purity of 99%). Procyanidin B3 Synthesis. Procyanidin B3 was obtained by hemisynthesis between (+)-taxifolin and (+)-catechin.12 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 Ar for 10 min. A sodium borohydrate solution previously degassed under argon was dropped under not oxidized 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 by Toyopearl gel column according to previous experiments.13 The B3 obtained had a purity >90% determined by LC-MS. Saliva Collection and Treatment. Saliva was collected from three healthy nonsmoking volunteers, and they were named saliva A, saliva B, and saliva C. Triplicates were collected for each saliva sample. The saliva samples were mixed with 10% TFA (final concentration = 0.1%) to precipitate several high molecular weight salivary proteins (such as α-amylases, mucins, carbonic anhydrase, and lactoferrin) and to preserve sample protein composition (histatins, PRPs, and statherins), because TFA partially inhibits intrinsic protease activity. After centrifugation (8000g for 5 min), supernatant was separated from the precipitate and used for the following experiments. HPLC-UV Saliva Analysis. Saliva samples were analyzed by HPLC-DAD prior to mass spectrometry analysis according to previous studies.11,14 An HPLC-DAD Elite Lachrom system (L-2130) equipped with a Vydac C8 column, with 5 μm particle diameter (column dimensions 150 × 2.1 mm), was used. Detection was carried out at 214−280 nm, using a diode array detector (L-2455). The HPLC solvents were (eluent A) 0.2% aqueous TFA and (eluent B) 0.2% TFA in ACN/water 80:20 (v/v). The gradient applied was linear from 10 to 40% (eluent B) in 60 min, at a flow rate of 0.30 mL/min. After this first elution program, the column was washed with 100% eluent B for 20 min to elute S-type cystatins and other late-eluting proteins. After washing, the column was stabilized in the initial conditions. Mass Spectrometry Analysis. Mass spectrometry analysis was used to identify salivary proteins and to assess the tannin−protein aggregate formation. The method described as follows is based on that of a previous study.17 ESI-MS Analysis. An aliquot of each assay was injected by flow injection analysis (FIA) into an LTQ-OrbitrapXL mass spectrometer, from ThermoFisher Scientific (Waltham, MA, USA). Sample was pumped at 5 μL/min using nitrogen as sheath gas at a flow of 5 (arbitrary unit system). Electrospray conditions from 3 kV spray voltage, 270 °C capillary temperature, and tube lens are established at 100 V at optimal analysis conditions. Samples were diluted into a methanol/acetonitrile/ TFA 0.01% (5:5:90 v/v) mixture 1:10 prior to analysis. Xcalibur 2.2 software was used to control the analysis and to interpret the data obtained. Because proteins acquire different charge states in the interface, after the mass spectrometry analysis, spectra were subjected to a deconvolution process using the charge ratio analysis method (CRAM) by MagTram 1.03 software. MALDI-TOF Analysis. One microliter of saliva or tannin−saliva sample (dilution 1:10) was applied onto a stainless steel target plate (MTP 394 target plate Ground steel BC, BrukerDaltonik GmbH), overlaid with 1 μL of matrix solution containing 10 mg/mL sinapic acid in acetonitrile, water, and TFA (50:47.5:2.5, v/v), and finally air-dried. Samples were spotted in triplicates. Mass spectra were automatically acquired on an UltrafleXtream MALDI-TOF-TOF mass spectrometer (BrukerDaltonics) operating in linear positive ion detection mode with laser SmartBeam-III and under FlexCompass 1.4 software control (BrukerDaltonics). For each sample the mass spectra (range from 500 to 50 000 Da) of 1000 laser shots were accumulated with 2000 Hz frequency. B3−Human Salivary Protein Aggregation. Three different salivas (A, B, and C) were mixed with procyanidin B3. The differences in salivary protein profiles between samples were evaluated by HPLC.
Further aggregation was studied by mass spectrometry (ESI-MS and MALDI-TOF). This experimental study was designed to establish differences in the aggregation process between salivas with different protein profiles and tannin at different concentrations. Differences in B3−salivary protein aggregates were established at two tannin concentrations (0.5 and 1 mg/mL procyanidin B3). Saliva A and saliva C were treated with 0.5 and 1 mg/mL B3, respectively, whereas saliva B was treated with both B3 concentrations. For all assays, 50 μL of B3 was added to 150 μL of saliva sample. After vortexing for 5 min, the samples were maintained for 30 min at room temperature prior to analysis. Kinetics Stability of Tannin−Protein Aggregates. Fifty microliters of B3(1 mg/mL) was added to 150 μL of saliva C to study the aggregates tannin−salivary protein and follow their evolution in time after 1, 6, and 20 h. All binding assays were analyzed in triplicates both by MALDI-TOF and by ESI-MS. Results obtained by FIA-ESI-MS were submitted to a deconvolution process. Blank subtraction with saliva control was made prior to the introduction of data into the MagTram software. Thus, the information obtained by ESI-MS was simplified, and the tannin−salivary protein complex deconvolution mass spectra became clearer. Phyton is a program language that is used in a wide variety of application domains, often in mass spectrometry to compare or handle spectra.15 It was employed as an identification bioinformatic tool to elucidate the structure of new tannin−protein aggregates. In this case, Phyton was used to reckon the different links between the different tannins assayed and the salivary proteins. This software works by matching the new m/z values with the different protein m/z values present in the control and each tannin added m/z value. Adducts and loss of ions such as H3O+ or NH4+ as well as reduction or oxidation processes have been considered. After running Python, several matches were hypothesized for each assay. Only the aggregate identifications matched by the two analytical techniques used (MALDI-TOF and ESI-MS) were considered. MALDI-TOF sensitivity decreased as higher analysis ratios were used. When B3−salivary protein aggregate stability was evaluated over time, higher aggregates were formed; thus, this phenomenon was worsened. ESI-MS was, therefore, the main technique chosen to discern the evaluation of aggregates formed over time. Statistical Treatment. All data values obtained empirically were treated statistically to observe significant differences. Graphpad Prism 6.1 was the software used in data statistical treatment. The Tukey test was used to establish differences in a single-step multiple comparison (it compares every mean with every other mean). The Tukey test gave a more precise value for scatter (mean square of residuals), which was reflected in more degrees of freedom. When mean A is compared to mean C, the test compares the difference between means to the amount of scatter, quantified using information from all of the groups, not just groups A and C. This gives more power to detect differences and made sense under the assumption that all of the data are sampled from populations with the same standard deviation, even if the means were different.
Figure 1. Salivary protein chromatographic profile from three salivas (A, B, and C). bPRPs, basic proline-rich proteins; gPRPs, glucosylated proline-rich proteins; His, histatins; aPRPs, acidic proline-rich proteins; Stath, statherins. 10039
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Figure 2. MALDI-TOF B3−human salivary protein aggregates profile. aPRP, acidic proline-rich protein; PRP3diphosphate, proline-rich protein 3 diphosphate; PRP1diphosphate, proline-rich protein 1 diphosphate; gPRP, acidic proline-rich protein; IB1, glucosidic proline-rich peptide IB1; II-2phosphate, glucosidic proline rich peptide II-2phosphate; IB8c, basic proline-rich peptide IB8c; IB8b, basic proline-rich peptide IB8b; PJ, basic proline-rich peptide PJ; IB6, basic proline-rich peptide IB6; IB9, basic proline-rich peptide IB9; IB5, basic proline-rich peptide IB5; IB4, basic prolinerich peptide IB4; His, histatins; His2, histatin 2; His 3, histatin 3; His 1, histatin 1; His 6, histatin 6; His 5, histatin 5; PB, statherin peptide PB; Statherin diphosp, Statherin diphosphate; Statherin monophosp, Statherin monophosphate; Statherin nphosp, Statherin n-phosphate. 10040
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RESULTS The aggregation between procyanidin B3 and human saliva (A, B, C) with different protein profiles was studied by mass spectrometry. Figure 1 shows the overlaid chromatographic protein profile (HPLC-DAD) of salivas A, B, and C. After HPLC-DAD analysis, some chromatographic differences were evident between the different salivas. The chromatographic protein profile was identified on the basis of previous studies.14 The different groups of proteins appeared characterized in the chromatogram by different parts according to their elution times. The first eluted peaks constituted the bPRPs (20−30 min), the second eluted peaks corresponded to gPRPs (30−35 min), the third chromatographic zone corresponded to aPRPs (45−50 min), and the last one contained the statherins (Stath) (60−65 min). Histatins (His) eluted at different retention times along the chromatogram, but they are mainly embedded at times around 40 and 55 min.12,14 The salivary protein content is described in Table 1 for all samples analyzed. Several quantitative changes were observed Table 1. Percent Area of Salivary Proteins (PRPs, Statherins, and Histatins) from Three Different Saliva Samples (A, B, and C) PRPs
bPRPs gPRPs aPRPs
histatins statherins total salivary proteins (referred to saliva Ba)
saliva A
saliva B
saliva C
18.31 14.75 33.73 9.66 23.55 10.25
39.27 31.93 18.34 2.73 7.73 100
37.15 31.97 19.97 1.59 9.32 75.44
a
Saliva B was considered as being 100% due to its highest chromatographic area.
between saliva samples. The salivary protein content was expressed as relative percentage area values. To compare the relative protein concentrations between saliva samples, saliva B was considered as being 100% due to its highest chromatographic peak area. Significant differences in the salivary proteins were observed between samples. Saliva A and saliva C have relative amounts of 10.25 and 75.44% of saliva B, respectively. In saliva A, the aPRPs comprised the group of salivary protein with higher area values followed by statherins, bPRPs, gPRPs, and histatins. However, in saliva B and C, the bPRPs was the salivary protein with higher area followed by gPRPs, aPRPs, statherins and histatins (Table 1). The soluble B3−salivary protein aggregates formed with these three saliva samples were characterized by mass spectrometry. MALDI-TOF was used to identify the B3−salivary protein spectra profile, and all data results were confirmed by ESI-MS. Forty-eight soluble B3−protein aggregates were identified (Supporting Information Table S1). The major soluble B3− protein aggregates according to the MALDI-TOF spectrum profile for saliva B are shown in Figure 2. Twenty-three major B3−salivary protein aggregates and some adducts are shown in the mass spectra. The majority of B3−salivary protein aggregates are embedded in the m/z 8000−16000 range (1−13 aggregates) and the m/z 20000−22000 range (14−23 aggregates). The majority of aggregates had His in their composition (mass peaks 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 19, and 22). Mass peaks 1, 4, 7, 10, 14, 20, 22, and 23 had bPRPs in their composition, and mass
Figure 3. B3−salivary protein aggregates comparison between different salivas at different tannin concentrations: number and molecular mass of aggregates (A); protein/tannin ratio (B); number of proteins or tannins involved in aggregate formation (C).
peaks 5, 10, 12, 13, 15, 17, 19, and 23 were formed by Stath. aPRPs and gPRPs are supposed to be part of only two major aggregates, 18 and 21 and 2 and 23, respectively. Figure 3A shows the differences of number and molecular mass of all soluble B3−salivary protein aggregates identified after statistical data treatment. All aggregates had molecular masses in the range from 8000 to 25000 Da. The median molecular mass of soluble B3−salivary protein aggregates formed was the same regardless of the saliva sample and tannin concentration tested (≈11000 Da). Nevertheless, little differences can be observed. At higher B3 concentration, saliva B yielded B3−salivary protein aggregates with higher molecular weight mass range than saliva C except the three aggregates (symbolized by points) 10041
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Figure 4. B3−salivary protein aggregates between different salivas at different tannin concentrations: occurrence of PRPs (A) and histatins and statherins (B). aPRP, acidic proline-rich protein; PRP3diphosphate, proline-rich protein 3 diphosphate; PRP1diphosphate, proline-rich protein 1 diphosphate; gPRP, acidic proline-rich protein; IB1, glucosidic proline-rich peptide IB1; II-2phosphate, glucosidic proline-rich peptide II-2phosphate; IB8c, basic proline-rich peptide IB8c; IB8b, basic proline-rich peptide IB8b; PJ, basic proline-rich peptide PJ; IB6, basic proline-rich peptide IB6; IB9, basic proline-rich peptide IB9; IB5, basic proline-rich peptide IB5; IB4, basic proline-rich peptide IB4; His, histatins; His2, histatin 2; His 3, histatin 3; His 1, histatin 1; His 6, histatin 6; His 5, histatin 5; PB, statherin peptide PB.
that appeared disperse with a molecular mass ≥20000. At lower B3 concentration, no differences were observed in the size of aggregates formed. Furthermore, no difference was observed in the molecular mass of the aggregates identified when two different tannin concentrations were tested (saliva B). Even though little or no differences were observed in the molecular mass, several differences were observed in the amount of aggregates identified. Forty-four aggregates were identified in saliva B, whereas saliva A yielded only 28 soluble B3−salivary protein aggregates at low tannin concentration. At higher tannin concentration, saliva B yielded 32 soluble B3−salivary protein aggregates and 26 were identified in saliva C. The differences in protein/tannin ratio depending on the saliva or tannin concentration tested are shown in Figure 3B. At lower tannin concentration (0.5 mg/mL B3), there were no significant differences in protein/tannin ratio of soluble B3−salivary protein aggregates regardless of the saliva tested. Nevertheless, at higher tannin concentration (1 mg/mL B3) the median protein/tannin ratio of soluble B3−salivary protein
aggregates yielded with saliva C was lower than those obtained with saliva B. Moreover, at higher B3 concentration, the range of protein/tannin ratio was higher for saliva B. The protein/tannin ratio reached values rounding 4, whereas saliva C did not achieve a value of 2. The protein/tannin ratio of soluble B3−salivary protein aggregates tended to be higher with higher tannin concentration when the same saliva was tested (Figure 3B). As described in Figure 3C, the number of tannin and protein moieties involved in soluble B3−salivary protein aggregate formation at lower B3 concentration was almost the same. At higher tannin concentration (1 mg/mL), the number of protein moieties was higher than the number of B3 units in the aggregate formation either in different salivas or in the same saliva. As aforementioned, the major aggregates had His in their composition, followed by bPRPs. Figure 4 shows the occurrence of salivary proteins in soluble B3−salivary protein aggregates depending on the saliva and/or tannin concentration used. No differences were found in salivary proteins that yielded the majority of soluble B3−salivary protein aggregates either with different 10042
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At 6 h their size increased to a median of 18000 Da, and it further decreased to 13000 Da after 20 h of incubation. Although the median molecular mass followed a path first decreasing, increasing, and then decreasing once again, the trend was to form higher aggregates with time, but these aggregates appeared statistically scattered (symbolized by points) (Figure 5A). It should be noted that after 6 h, slight turbidity was observed in samples, probably resulting from the formation of insoluble complexes, and the homogeneity of samples was lost. The number and molecular mass of aggregates has evolved over time, leading to new B3−human salivary protein aggregates, but some stable complexes were maintained over time. After 6 h of incubation time, six B3−salivary protein complexes did not evolve, appearing at the same signal spectra; they are the B3− statherin n-phosphate, 6B3−His6, B3−IB9, 3B3−statherin n-phosphate, 5B3−statherin monophosphate, and 4B3−PJ. At 20 h only two aggregates were maintained, the B3−IB9 (m/z 6623) and 3B3−statherin n-phosphate (m/z 6992). On the other hand, the trend after 6 h was for major aggregates to be composed of more than five proteins and only one or two tannin moieties. The median protein/tannin ratio of B3−salivary protein aggregates was maintained from control to 1 h, then it increased to a maximum median value at 6 h, and finally decreased after 20 h of incubation (Figure 5B). These differences in protein/tannin ratio of B3−salivary protein aggregates are given by the change in protein moieties involved in B3−salivary protein aggregate formation among time (Figure 5C). As Figure 5C shows, the trend is to increase the number of protein moieties involved in aggregation process from control to 1 and 6 h, and then the number of protein moieties involved in B3−salivary protein aggregates was maintained until 20 h of incubation. On the other hand, the number of tannin moieties involved in aggregation changed only at 6 h, decreasing to a median value of 1 per soluble aggregate. Figure 6 shows the scrutiny of proteins involved in aggregate formation. From control to 1 h no difference was observed in the scrutiny of proteins involved in the aggregate formation. The protein group with higher occurrence in B3−salivary protein aggregates was His, followed by bPRPs, Stath, gPRPs, and aPRPs. After 6 h, His are no longer the group of proteins with higher occurrence in the soluble B3−human salivary protein aggregates. The bPRPs became the group of proteins that yielded higher number of aggregates followed by Stath. Furthermore, most of the aggregates were only formed, in descending order, by IB8b, His6, PRP3diphosp, Stath monophosphate, Stath n-phosphate, IB9, peptide PB, and peptide PJ. After 20 h, most of the aggregates were formed by the same proteins but the order was altered. The proteins involved in higher number of aggregates after 20 h were, in descending order, PB, PJ, His6, IB9, Stath monophosphate, IB8b, Stath n-phosphate, and PRP3diphosp, respectively.
saliva or with different tannin concentrations. The protein group with higher occurrence in soluble B3−salivary protein aggregates was the His followed by bPRPs, Stath, gPRPs, and aPRPs. Time Stability of Soluble B3−Human Salivary Protein Aggregates. Aggregation between B3 and salivary proteins was studied by mass spectrometry analysis (ESI-MS and MALDITOF) over time. MALDI-TOF and ESI-MS spectra showed trending for new mass peaks to appear at higher molecular mass range (data not shown). The aggregates formed during reaction time are described in Table S2 in the Supporting Information. Figure 5 shows the evolution over time in terms of number and
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Figure 5. B3−salivary protein aggregate stability over time: number and molecular mass of aggregates (A); ratio protein/tannin (B); number of proteins or tannins involved in aggregate formation (C).
DISCUSSION Individual chromatographic differences between samples were verified from salivas A, B, and C. This fact could influence differences in the B3−salivary protein aggregation process at the two tannin concentrations tested (0.5 and 1 mg/mL). The median molecular mass rounded 11000 Da regardless of the saliva or tannin concentration used. Several differences were found in the number of aggregates formed at different tannin concentrations and saliva samples tested. In general, it could be assumed that the number of soluble aggregates formed was higher at lower B3 concentration. On the other hand, a higher number of aggregates was observed at higher salivary protein
molecular mass of the aggregates formed, protein/tannin ratio, and the scrutiny of proteins and tannin moieties involved in the formation of B3−human salivary protein aggregates. After 1 h of incubation time at room temperature, the number of aggregates found in saliva C increased from 26 to 42; it further increased exponentially after 6 h, leading to a maximum of 166 B3−human salivary protein aggregates, and it went down to 130 at 20 h. The median of molecular mass of aggregates also experienced some changes over time. Most of the aggregates decreased their size from 11000 to 7500 Da during the first hour of incubation time. 10043
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Figure 6. B3−salivary protein aggregates stability over time: occurrence of PRPs (A) and histatins and statherins (B). aPRP, acidic proline-rich protein; PRP3diphosphate, proline-rich protein 3 diphosphate; PRP1diphosphate, proline-rich protein 1 diphosphate; gPRP, acidic proline-rich protein; IB1, glucosidic proline-rich peptide IB1; II-2phosphate, glucosidic proline-rich peptide II-2phosphate; IB8c, basic proline-rich peptide IB8c; IB8b, basic proline-rich peptide IB8b; PJ, basic proline-rich peptide PJ; IB6, basic proline-rich peptide IB6; IB9, basic proline-rich peptide IB9; IB5, basic prolinerich peptide IB5; IB4, basic proline-rich peptide IB4; His, histatins; His2, histatin 2; His 3, histatin 3; His 1, histatin 1; His 6, histatin 6; His 5, histatin 5; PB, statherin peptide PB.
concentration (area values). The results presented herein also confirmed that for the same saliva sample at higher tannin concentration, the number of protein moieties involved in B3−salivary protein aggregate formation was also higher. These results are in agreement with previous studies. Cannon et al.8
verified that at lower tannin concentrations at least, half of the protein added did not bind to tannin. According to Lorenz et al.,16 increasing the tannin concentration decreases the protein solubility, so this fact could complicate the formation of a higher number of aggregates. 10044
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The protein and/or tannin selectivity was also verified regardless of the saliva samples and tannin concentration tested. The same group of salivary proteins yielded the higher number of aggregates despite some salivary protein chromatographic differences between samples. These results are in agreement with previous studies because His was the group of proteins that produced a higher number of aggregates when saliva was treated with B3.17 Although His was the group of proteins that formed a higher number of soluble B3−salivary protein aggregates studied by mass spectrometry, it should be pointed out that Stath and aPRPs were the more reactive groups of proteins and led to a higher precipitation to form insoluble complexes.14,18 Stability over time was also studied showing that the B3−salivary protein aggregates evolved in time. After 1 h of incubation, the trend was to decrease the molecular mass of the B3−salivary protein aggregates. It is well-known that protein− tannin noncovalent aggregation is a reversible process in aqueous models.19−21 After 1 h, new B3−salivary protein aggregates were formed, whereas the first B3−salivary protein aggregates were broken up. The trend after 6 h within the following 20 h was to increase the number of protein moieties involved in the aggregation process, and only a few aggregates were maintained. These results are in agreement with previous studies in which the number of protein moieties forming aggregates increased over time.8 On the other hand, it should be noted that the group of proteins involved in aggregates changed over time. Whereas His was the protein group with higher selectivity from the initial aggregation process to 1 h, after 6 h, the bPRPs and Stath yielded the higher number of aggregates. Hence, the B3−salivary protein aggregates changed not only in number but also in the type of proteins involved in their composition. The results presented herein highlight the effect of the tannin concentration in the formation of different soluble B3−salivary protein aggregates. Furthermore, the individual effect of the different salivas has been exhibited. The differences in B3−salivary protein aggregates were based primarily in the number of complexes formed and hence not their molecular masses. Moreover, the protein/tannin ratio barely varied between different saliva samples or tannin concentration, but the number of tannin moieties involved in the aggregation process was higher at higher B3 concentrations. On the other hand, the evolution of B3−salivary protein was revealed because a higher number with higher size was formed and their stoichiometry also changed over time.
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REFERENCES
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ASSOCIATED CONTENT
S Supporting Information *
FIA-ESI-MS spectra of human saliva protein and B3−human saliva protein complexes as well as MALDI-TOF spectra accuracy and sensibility. This material is available free of charge via the Internet at http://pubs.acs.org.
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Article
AUTHOR INFORMATION
Corresponding Author
*(V.D.F.) Phone: +351 220 402 558. Fax: 351 220 402 659. E-mail:
[email protected]. Funding
We thank the Fundacaǫ para a Ciencia e Tecnologia (FCT), which funded this work by a postdoctoral fellowship (SFRH/ BPD/85293/2012) and by a research project grant (NORTE07-0162-FEDER-000048). Notes
The authors declare no competing financial interest. 10045
dx.doi.org/10.1021/jf5033284 | J. Agric. Food Chem. 2014, 62, 10038−10045
