Protein–Phenolic Interaction of Tryptic Digests of β-Lactoglobulin and

Article pubs.acs.org/JAFC

Protein−Phenolic Interaction of Tryptic Digests of β‑Lactoglobulin and Cloudberry Ellagitannin Bei Wang, Tuuli Koivumak̈ i, Petri Kylli, Marina Heinonen,* and Marjo Poutanen Department of Food and Environmental Sciences, Division of Food Chemistry, University of Helsinki, P.O. Box 27, FI-00014 University of Helsinki, Finland ABSTRACT: LC-ESI-MS was applied to investigate interaction reactions between a dimeric ellagitannin, sanguiin H-6, isolated from cloudberries (Rubus chamaemorus) and peptides of β-lactoglobulin (β-Lg). Three peptides, LIVTQTMK (m/z 934), ALPMHIR (m/z 838), and IPAVFK (m/z 674) were isolated from enzymatic (trypsin) digestion of β-Lg. Oxidation of the peptides with and without sanguiin H-6 was monitored by LC-ESI-MS for up to 7 days. Sanguiin H-6 showed radical scavenging activities toward oxidation of the selected peptides. An interaction product was found with sanguiin H-6 and peptide LIVTQTMK by using MS and supported by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). An observable (haze) but unstable interaction product of sanguiin H-6 was seen with peptide ALPMHIR, but no detectable interaction products were seen with peptide IPAVFK. A higher proportion of sanguiin H-6 toward the amount of peptide might allow for further characterization of these interaction products. KEYWORDS: β-lactoglobulin, peptides, sanguiin H-6, ellagitannin, digestion, LC-ESI-MS

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system.9,10 The ellagitannin monomers tend to form dimers, trimers, and even higher oligomers via phenolic oxidative coupling reactions. In raspberries (Rubus idaeus L.), the major ellagitannins have been identified as the dimeric sanguiin H-6 (Figure 1) and the trimeric lambertianin C. The ellagitannin

INTRODUCTION Bovine β-lactoglobulin (β-Lg) is an important whey protein in bovine milk comprising up to 50−55% of total whey proteins. It is mostly present in ruminant species, such as sheep, horse, reindeer, and in pigs. Therefore, it widely exists in milk-based food products. The nutritional value, potential biological functions, and allergenicity of β-Lg are well-known. β-Lg is resistant to enzymatic digestion, particularly to chymotrypsin and pepsin; however, it is susceptible to trypsin.1 β-Lg contains 162 amino acids with cysteine, methionine, histidine, lysine, threonine, arginine, proline, tryptophan, tyrosine, and phenylalanine being the most potential amino acids considered to be oxidized.2,3 According to Elias et al.,4,5 βLg is more stable in its intact form than its enzyme-hydrolyzed peptide fragments. Methionine in peptide SLAMAASDISLL (positions of 11−20) and tyrosine in peptide DIQKVAGTWY (positions of 21−32) are the most potential oxidized residues in β-Lg.4,5 Tryptophan, threonine, methionine, and histidine were proved to have oxidative tendency due to their high hydrogendonating ability.6 Cysteine in the position of 121 has mild antioxidant potency due to the only free sulfhydryl group in the protein, which can act as hydrogen donor.7 In the study of Koivumäki et al.,8 three tryptic peptides of β-Lg prone to oxidize and dioxidize were chosen to investigate the oxidation of β-Lg. These peptides were alanine-leusine-proline-methionine-histidine-isoleucine-arginine (ALPMHIR), leucine-isoleucine-valinethreonine-glutamine-threonine-methionine-lysine (LIVTQTMK), and valine-leucine-valine-leucine-asparaginethreonine-aspartic acid-tyrosine-lysine (VLVLDTDYK). The promising indicators of β-Lg oxidation were reported to be the ions m/z 854 [ALPMHIR +O], m/z 950 [LIVTQTMK + O], and m/z 966 [LIVTQTMK + 2O]. Berry phenolics including tannins such as ellagitannins and proanthocyanidins were discovered to be effective in quenching protein oxidation in the α-lactalbumin-lecithin liposome © 2014 American Chemical Society

Figure 1. Structure of sanguiin H-6 dimer with indicating groups of glucose, galloyl, and HHDP.

composition of cloudberries (Rubus chamaemorus L.) is less studied apart from McDougall et al.11 and Kähkönen et al.12 reporting lambertianin C as the predominating ellagitannin. In 1966, Loomis and Battaile13 proposed that phenols may reversibly complex with proteins through hydrogen bonding or irreversibly by oxidation to quinones that combine with reactive groups of protein molecules. Recent studies prove that phenolic compounds may interact with protein through both covalent Received: Revised: Accepted: Published: 5028

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carried out by using preparative HPLC and assigning the automated collection triggers for the corresponding m/z values of the theoretical peptides. Isolation of Sanguiin H-6. Freeze-dried cloudberries (2 g, powder) were extracted with 70% acetone (2 × 20 mL) by accelerated solvent extraction (ASE) (Dionex, Sunnyvale, CA, U.S.A.). The extracts were evaporated to dryness and dissolved into Milli-Q water. Column (40 × 300 mm) chromatography with Amberlite XAD-7 was used for removal of sugars, organic acids, and phenolic acids eluting with 6% acetonitrile. A fraction containing flavonols, anthocyanins, and ellagitannins was collected using 100% acetonitrile. The dried polyphenol fraction dissolved into aq methanol (50%) was subject to further fractionation using a Sephadex LH-20 column (40 × 300 mm). The ellagitannin fraction was eluted with 70% acetone. An ellagitannin dimer, sanguiin H-6, was obtained from the ellagitannin fraction by using preparative-HPLC (Waters 2767 sample manager, Waters 2545 binary gradient module, and Waters system fluidic organizer) coupled with a Waters 2998 PDA and Waters ZQ single quadrupole mass spectrometer (Waters, Milford, MA, U.S.A.). The column was Develosil ODSHG-5 column, 250 × 20 mm, 5 μm (Phenomenex, Torrance, CA, U.S.A.). The mobile phase consisted of 0.1% formic acid in water (solvent A) and 50% acetonitrile in water (solvent B). The elution condition was as follows: isocratic elution 0% B 0−2 min; linear gradient from 0% to 6% B, 2−8 min; from 6% to 10% B, 8− 25 min; from 10% to 16% B, 25−40 min; isocratic 16% B 40−50 min; linear gradient from 16% to 64% B, 50−53 min; isocratic 64% B, 53−58 min; linear gradient from 64% to 0% B, 58−0 min. Injection volume was 1.0 mL. The flow (5.0 mL/min) was split 1:100 before the PDA and mass spectrometry (MS) detection. PDA scan range was 210−600 nm. Mass spectrometry (MS) was fitted with an ESI source and was operated in negative mode. The capillary voltage was 3.9 kV, cone 66.00 V, extractor 3.00, and RF lens 0.7 V. Source temperature was 120 °C, desolvation temperature 350 °C, cone gas flow 50 L/h, and desolvation gas flow 600 L/h. The scan range of the mass spectrometry was m/z 1200−2000. The m/z value 1869 (sanguiin H-6 [M-H]−) was used as a trigger for the fraction collection. The system was controlled by Waters MassLynx software version 4.1 with FractionLynx (Waters). The purity and identity of the isolated sanguiin H-6 was determined using the UHPLC-PDA-MS method outlined by Kähkönen et al.,12 and the concentration was determined spectrophotometrically using the molar extinction coefficient ε260 nm 72070 M−1cm−1 for sanguiin H-6 in methanol.30 The concentration of isolated sanguiin H-6 was 254.3 μg/mL in methanol with a purity of 96%. Oxidation of the Peptide Samples With and Without Sanguiin H-6. The three chosen peptides, LIVTQTMK, ALPMHIR and IPAVFK, were prepared into separate oxidation samples in triplicates, as well as both with and without the sanguiin H-6. The phenolic-to-peptide ratio was chosen to be 1000 μg of sanguiin H-6/g of peptide (molar ratio of 1:2000) according to pretesting done with LIVTQTMK, ALPMHIR, and IPAVFK. For further studies regarding the nature of the adduct between peptide and the ellagitannin, 162 mg sanguiin H-6/g of peptide (molar ratio of 1:12.5) was used with LIVTQTMK. After sanguiin H-6 was added to samples, H2O2 solution (final concentration 1 mM) was added into each sample just before the reaction in order to start the oxidation reactions. The samples were placed in an oven of +37 °C for 7 days with stirring on a magnetic tray. An aliquot of 200 μL of each sample was taken on

links and noncovalent links. Noncovalent interaction includes hydrogen bonding, hydrophobic bonding, and sometimes even ionic bonding. It occurs depending on the structure of polyphenols and various functional groups in proteins.14−20 Noncovalent interaction could take place multisitely and multidentately due to the polarity of the polyphenol. Multisite interaction means that several phenolic compounds bound to one protein molecule, whereas multidentate interaction means that one phenolic compound bound to several protein sites or molecules. The type of interaction relies on the type and the molar ratio of both phenolic compound and protein.21,22 Covalent interaction occurs through the interaction between oxidized phenolic substances and proteins.23,24 Electrospray ionization (ESI) has been proven to be an effective tool to detect the weak associations of molecules in solutions such as receptor− ligand and protein−nucleic acid interaction.25,26 A relatively welldeveloped way to determine the relative binding affinities of noncovalent complex has been established.27,28 The mass spectrometry (MS) can reveal the noncovalent interaction strengths and qualitative information on the complex structure. Therefore, MS instrumentation was also used to detect and characterize soluble noncovalent complexes.29 In this study, the aim was to apply LC-ESI-MS to investigate the radical scavenging and other protein−phenolic interaction reactions between cloudberry ellagitannin and tryptic digests of β-Lg. Tryptic peptides of β-Lg have been used as a model to study protein−phenolic interactions. This simplified model enables the mechanistic investigations in more detail compared to those of a complicated protein. This model mimics possible reactions between proteins or peptides and plant phenolics during in vivo digestion but also in the food matrix as protein binding would affect polyphenol reactivity and influence its biological fate.

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MATERIALS AND METHODS Chromatographically purified and lyophilized β-Lg (variants A + B from bovine milk) was purchased from Sigma-Aldrich, Inc. (St. Louis, MO, U.S.A.). Sequencing-grade modified trypsin was obtained from Promega Corp./BioFellows (Madison, WI, U.S.A.). Of the reagents used in the analyses, ammonium bicarbonate, hydrogen peroxide (30% wt solution in water), and iron(III) chloride (reagent grade, 97%) were purchased from Sigma-Aldrich (Steinheim, Germany), whereas L(+)-ascorbic acid was a product of Merck (Darmstadt, Germany) and PIPESbuffer [piperazine-1,4-bis(2-ethanesulfonic acid)] from Fluka BioChemika (Buchs, Switzerland). Methanol, acetone, and acetonitrile were of HPLC grade and purchased from Rathburn (Walkerburn, Scotland). All other chemicals used in the analyses were supplied by J. T. Baker (Deventer, The Netherlands) or Sigma-Aldrich in either HPLC or reagent grade. The water used was always purified first by the Milli-Q system (Millipore Corp., Bedford, MA, U.S.A.). Fresh cloudberries (Rubus chamaemorus) were purchased from the local grocery. Digestion and Fractionation of β-Lg Peptides. The inliquid digestion of β-Lg was prepared using sequencing grade modified trypsin to obtain the cleavage to peptides which were later separated and collected using preparative HPLC according to the method described by Koivumäki et al.8 In brief, 10 mg of βLg was dissolved in 50 mM ammonium bicarbonate buffer followed by addition of trypsin with a protease/protein ratio of 1:250. The protein digest was kept at 37 °C overnight, and thereafter the enzyme reaction was stopped by placing the digest in −20 °C. Isolation and fractionation of β-Lg peptides was 5029

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Figure 2. Full scan mass spectrum of isolated sanguiin H-6.

Figure 3. Extracted ion chromatograms (EIC) of the peptide LIVTQTMK ([M + H]+ m/z 934), Met sulfoxide ([LIVTQTMK + O]) form of peptide LIVTQTMK, and Met sulfone ([LIVTQTMK + 2O]) form of peptide LIVTQTMK in positive mode by MS from day 0.

Santa Clara, CA, U.S.A.), which were all connected to a Bruker Esquire quadrupole ion trap mass spectrometer (QIT-MS, Bremen, Germany) using ESI in both positive and negative mode. The column was a Waters XBridge BEH130 C18 (3.5 μm, 2.1 × 100 mm) together with a precolumn (both by Waters Corp., Wexford, Ireland). Injection volume was always 10 μL, and the column temperature was kept at +30 °C. Flow rate was 350 μL/min, and the eluents consisted of 0.1% formic acid in

days 0, 1, 4, and 7 for analysis of reaction products. The sub samples were collected into Eppendorf tubes and immediately frozen at −20 °C until analysis. Analysis of the Oxidation and Interaction Products by LC−MS. The LC−MS used in the analysis of all the oxidation samples was an Agilent 1100 HPLC including a binary pump, a degasser, an automated sample manager, a column heating unit and DAD, and fluorescence detectors (Agilent Technologies, 5030

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water (solvent A) and 0.1% formic acid in acetonitrile (solvent B). The diode array detector was set to record at 214 nm, and the fluorescence detector was set to 280 nm (Ex) and 350 nm (Em). The gradient used as well as all the MS-parameters for the positive mode were consistent with the method presented by Koivumäki et al.8 Only in the negative mode the MS-parameters were optimized using peptides LIVTQTMK, ALPMHIR, and IPAVFK, and they were set to following: dry temperature +300 °C, dry gas 8.0 L/min, nebulizer 60.0 psi, capillary 3800 V, end plate offset −500, trap drive 102.5, capillary exit −178.5, lens-1 5.0, lens-2 60.0, and octopole rf amplitude 137.7 Vpp. For both positive and negative mode, the mass spectra were recorded in the full-scan mode over the m/z range of 200−2200 and analyzed by Bruker Daltonics Data Analysis software (Bremen, Germany). Fragmentation of Sanguiin H-6 and Interaction Sample. Pure sanguiin H-6 sample and the sample with 162 mg of sanguiin H-6/g of peptide were also analyzed by LC-MS in negative mode. DAD and MS parameters were the same as mentioned previously, except that the nebulizer was changed to 70 psi. The m/z scan range of pure sanguiin H-6 sample was 200−2200, and it was 100−1200 for day 1 oxidation sample with 162 mg of sanguiin H-6/g of peptide. Analysis of the Oxidation and Interaction Products by Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE). Peptide and peptide-sanguiin H-6 complex (peptide-SH6) were analyzed with SDS-PAGE. Samples contained 10 μL of concentrated peptide or peptideSH6 and 30 μL of sample buffer, which contained 12% SDS, 150 mM Tris/HCl pH 6.8, 0.05% bromophenol blue, and 30% glycerol, and they were incubated for 15 min at 37 °C and applied on gel wells.30 The gels were Mini Protean Tris-Tricine Precast gels, 10−20% with 50 μL wells (Bio-Rad, Hercules, CA, U.S.A.). Molecular weight standard, 1.5 μL, was applied on two separate wells (Thermo Fisher, 10−170 kDa). Electrophoresis was performed using a Mini protean Electrophoresis system (BioRad) at 20 °C as follows: maximum current throughout the run, 30 min at 30 V, 10 min at 60 and 100 V, until the electrophoresis was stopped. The anodic buffer was 1.0 M Tris and 0.225 M HCl at pH 8.9, and the cathodic buffer was 1.0 M Tris, 1.0 M Tricine, and 1.0% SDS at pH 8.25.31 After the electrophoresis, the gel was fixed overnight in 30% ethanol, 0.5% acetic acid in deionized water and stained with silver.32

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Figure 4. Changes of the amounts of peptide LIVTQTMK and its oxidation products in the samples with and without 1000 μg/g sanguiin H-6 and in positive mode detection (a) LIVTQTMK ([M + H]+ m/z 934), (b) [LIVTQTMK + O] ([M + H]+ m/z 950), and (c) [LIVTQTMK + 2O] ([M + H]+ m/z 966).

RESULTS AND DISCUSSION

MS Identification of Sanguiin H-6. A dimeric ellagitannin, sanguiin H-6, was isolated from cloudberry extracts and separated from the trimeric lambertianin C by using preparative HPLC. The isolated sanguiin H-6 was detected by MS in negative mode (Figure 2) with the fragmentation of m/z 1869. The fragment m/z 1869 of isolated sanguiin H-6 was further identified by MS/MS in negative mode. The retention time of m/ z 1869 was around 23 min. These data are in accordance with earlier findings.11,12 Hexahydroxydiphenoyl (HHDP), glucose, and galloyl groups are the basic functional groups contained in sanguiin H-6. The fragment ions of m/z 1567 (loss of HHDP), 1265 (loss of HHDP−HHDP), 1103 (loss of HHDP−HHDPglu), and 933 (loss of HHDP−HHDP-glu-galloyl) are the characteristics for sanguiin H-6.12 As the purity of isolated sanguiin H-6 was 96%, the rest of the extract contained peduculagin/casuarictin isomers because of the detection of m/z 1567, which was [2M − H]− of an ion m/z 783, potentillin/ casuarictin due to [M − H]− at m/z 935, and its further

Figure 5. SDS PAGE gel with wells 1 and 8 for molar weight markers, well 2 with day 0 peptide LIVTQTMK, well 3 with day 7 peptide LIVTQTMK, wells 4 and 5 with replicate samples of day 7 peptide LIVTQTMK with high concentration (with 162 mg of sanguiin H-6/1 g of peptide) sanguiin H-6, and wells 6 and 7 empty.

fragmentation at m/z 633 was observed in the mass spectra of isolated sanguiin H-6 extracts (Figure 2).33−35 Oxidation Products of Peptide LIVTQTMK and Its Interaction with Sanguiin H-6. Methionine sulfoxide (m/z 950) and methionine sulfone (m/z 966) forms of peptide LIVTQTMK were already observed in positive mode on day 0 (Figure 3), as previously reported by Koivumäki et al.8 The amount of the unoxidized peptide LIVTQTMK (m/z 934) on day 0 was higher in the samples containing sanguiin H-6 5031

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Figure 6. Day 0 LC-MS chromatograms in negative mode: (a) TIC of peptide LIVTQTMK without sanguiin H-6 and (b) TIC of peptide LIVTQTMK with 1000 μg/g of sanguiin H-6.

sanguiin H-6 interacts with ß-Lg with forming adducts this prevents the peptide from undergoing oxidation. The gel electrophoresis showed the peptide LIVTQTMK on day 7 with replicate samples of sanguiin H-6 (well 4 and well 5), with two lines on both, one around 170 kDa and the other between 55 and 72 kDa (Figure 5). The peptide LIVTQTMK sample also displayed one line between 55 and 72 kDa on day 0 (well 2), but none was detected on day 7 with the oxidized peptide sample (well 3). Because the molar weight of peptide LIVTQTMK was less than 10 kDa (the cutoff of the gel), the peptide LIVTQTMK alone in the sample cannot be detected. It is assumed that the masses around 170 kDa and possibly also around 55−72 kDa might be due to the interaction products between sanguiin H-6 and peptide LIVTQTMK. The interaction between peptide and sanguiin H-6 and the radical scavenging activity of sanguiin H-6 were observed by LCMS in the negative mode. Normally, the peptide is detected in positive mode by LC-MS, whereas some of the phenolic compounds such as ellagitannins can only be perceived in negative mode.12 The dimeric sanguiin H-6 with m/z of 1869 at [M − H]− in negative mode is shown in Figure 2. In the sample containing 1000 μg sanguiin H-6 per gram peptide LIVTQTMK, a new peak was found at retention time of 20 min compared with the sample without sanguiin H-6 on day 0 (Figure 6). The fragment ions m/z 1119 and m/z 997 that were detected on day 0 in negative mode may be the oxidation products of sanguiin H-6 fragments, which were m/z 1103 resulting in Δm = +16 amu (sanguiin H-6 loss of HHDP−HHDP-glu + O) and m/z 933

compared to the samples without sanguiin H-6 where the unoxidized peptide m/z 934 was not detectable (Figure 3). This may be due to the reduction of the methione sulfoxide form of peptide LIVTQTMK (by sanquiin H-6) back to the nonoxidized methionine residues in the peptide.36 In the presence of sanquiin H-6, the unoxidized peptide m/z 934 continued to decrease (Figure 4a), which may be partly due to adduct formation as the amount of oxidized peptide forms did not increase (Figure 4 b, 4 c). In samples without sanguiin H-6 (Figure 4c), more of the methionine sulfone form of the peptide (m/z 966) was formed from methionine sulfoxide (m/z 950) because of further oxidation. For all the three ions (m/z 934, 950, 966), the trends of ion change were much less significant with sanguiin H-6 than for those without (Figure 4). This might indicate a significant slow scavenging step with sanguiin H-6 present, which maintained the stability of the sample system.37 Rapid scavenging activity of sanguiin H-6 has earlier been seen toward lipid oxidation.12 Sanguiin H-6 has been reported to donate an electron to highly reactive free radicals and to suppress the reactivity of the radical by delocalization of the unpaired electron on the phenolic ring.38,39 Sanguiin H-6 might interact with protein through hydrogen bonding, hydrophobic bonding, or ionic bonding.18−20,40,41 For example, for hydrogen bonding, the carbonyl functional groups of the amino acids or peptide backbone and the isolated phenolic hydroxyl group in the polyphenolic form a strong hydrogen bond during protein− polyphenol interaction.15,42−45 It is postulated that when 5032

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Figure 7. Sanguiin H-6 and interaction sample. (a) TIC of sanguiin H-6. (b) Chromatogram of sanguiin H-6 with UV detection at 210 nm. (c) Chromatogram of sanguiin H-6 with UV detection at 280 nm. (d) Day 0 TIC of peptide LIVTQTMK with a high concentration (with 162 mg of sanguiin H-6/1 g of peptide) of sanguiin H-6. (e) Chromatogram of peptide LIVTQTMK with a high concentration (with 162 mg of sanguiin H-6/1 g of peptide) of sanguiin H-6 with UV detection at 210 nm. (f) Chromatogram of peptide LIVTQTMK with a high concentration (with 162 mg of sanguiin H-6/1 g of peptide) of sanguiin H-6 with UV detection at 280 nm.

Figure 8. TIC of peptide LIVTQTMK with a high concentration (with 162 mg of sanguiin H-6/1 g of peptide) of sanguiin H-6 from days 0, 1, 6, and 7. Peaks A and B refer to interaction products. 5033

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Figure 9. Peptide LIVTQTMK with a high concentration (with 162 mg of sanguiin H-6/1 g of peptide) from day 1 (a) TIC, (b) TIC of fragmentation of m/z 963.5, (c) MS/MS spectrum of peak 1, and (d) MS/MS spectrum of peak 2.

Compared to the MS/MS of peak 1 and 2 (Figure 10b), 877, 921, and m/z 947 in Figure 10c were 2 amu different compared with 875, 919, and m/z 945 in Figure 10d, which indicated there might be a double bond breakage. Furthermore, the 17 amu difference between m/z 921 and m/z 903 (Figure 9c), as well as m/z 902 and m/z 919 (Figure 9d), indicated that there might be an addition of ammonia. Lysine and methionine residues in peptide LIVTQTMK might be responsible for the interaction between peptide LIVTQTMK and sanguiin H-6. The interaction may occur owing to the hydrophoblic section in the side chain of lysine and methionine.18,46−49 In addition, lysine has a positive charge near neutral pH, which makes it possible to interact with the carboxylate group or the aromatic ring of sanguiin H-6. Oxidation Products of Peptide ALPMHIR and Its Interaction with Sanguiin H-6. Peptide ALPMHIR was detected with [M + H]+ m/z 838 in positive mode and [M − H]− m/z 836 in negative mode by LC-MS. Met, His, and Pro present in peptide ALPMHIR are rather prone to be monoxidized, whereas Pro, Arg, and Met tend to be dioxidized.2 [ALPMHIR + O] was observed to be the most prominent oxidation product formed during oxidation for 7 days, as earlier reported also by Koivumäki et al.8 Similarly to peptide LIVTQTMK ([M + H]+ m/z 934), during the oxidation process, the color of the peptide ALPMHIR ([M + H]+ m/z 838) samples changed from transparent to lightly yellowish with tiny milky-white particles dissolving out from time to time. The precipitations were probably due to instable haze formed due to interaction between peptide and sanguiin H-6.

with +64 amu addition (sanguiin H-6 loss of HHDP−HHDPglu-galloyl +4O). This suggests that the oxidation of sanguiin H6 itself happened quite rapidly on day 0. The detection of ion m/z 963.5 and its extracted ion chromatogram reveal that the interaction product between oxidized peptide and oxidized ellagitannin monomer (casuarictin) with doubly charged ions was generated on day 0. Even though the intensity of m/z 963 was relatively low, m/z 963 is very likely to be the interaction product between peptide LIVTQTMK and sanguiin H-6. The same peak indicating the formation of an interaction product was also detected at a retention time of 20 min (Figure 7d) in the samples containing a higher proportion of sanguiin H6 to that of peptide (162 mg of sanguiin H-6/1 g of peptide). The peak of sanguiin H-6 (Figure 7a) was not detected on day 0 in the sample of sanguiin H-6 with peptide LIVTQTMK, whereas a new peak appearing at a retention time of 20 min (Figure 7d) was also detected both at 210 and 280 nm. This indicates the presence of both peptide bond and phenolic group, supporting the conclusion that the new peak is an interaction product of sanguiin H-6 and peptide LIVTQTMK. Other peaks (with retention times between 14 and 24 min, Figure 8) were found in the total ion chromatogram (TIC) in the samples with a higher concentration of sanguiin H-6 with peptide LIVTQTMK. The areas of these six peaks were increasing from day 0 to day 7. Peak A disappeared after day 1, which could indicate that further oxidation occurred. Peak B, around 15 min in Figure 8, seems to be the main interaction product with covalent bonding, as it was also identified in Figure 9. This figure also shows the [M − H]− m/z 963, which was further fragmented to m/z 919 and m/z 875. 5034

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dioxidized form of peptide ALPMHIR (m/z 870), the amount of that in the samples with sanguiin H-6 was even higher (Figure 10c). This may be due to the dioxidation of proline, arginine, and methionine that occurred so rapidly and even faster than the scavenging activity of sanguiin H-6. Sanguiin H-6 may also have inhibited the further oxidation of dioxidized form of peptide ALPMHIR, and the amount of [ALPMHIR + 2O] was lower in the sample without sanguiin H-6, because many of them might be further oxidized. Contradictory to peptide LIVTQTMK, the sample without sanguiin H-6 was more stable than that with added sanguiin H-6 for peptide ALPMHIR. The reason might be the different amino acid composition of the peptides. Tannin presents both a fast and a significant slow scavenging step in the sample. Proline, methionine, histidine, lysine, and methionine are all capable to interact with sanguiin H-6. For example, proline residues could provide a hydrophobic binding site through pyrrolidine rings and may associate with aromatic rings in sanguiin H-6.18,46−48 Therefore, besides oxidations on different residues, phenolic−protein interaction, antioxidant, and scavenging activity of sanguiin H-6 were occurring together in the sample with sanguiin H-6. Thus, the amount of interaction products was less stable. The tiny milky-white precipitation particles dissolved out from time to time were likely to be the haze due to the interaction between peptide ALPMHIR and sanguiin H-6 because the peptide ALPMHIR contains proline, which is usually rich in haze-forming proteins.46 The haze was invisible to the eye until the formation of the polyphenol protein agglomeration precipitate. It was a less soluble entity most likely formed by the cross-linking of hydroxyl groups presented in polyphenol and protein molecules.50 Therefore, the presence of haze also indicated the existence of unstable interaction products. Oxidation Products of Peptide IPAVFK and Its Interaction with Sanguiin H-6. The peptide was detected at [M + H]+ m/z 674 in positive mode and [M − H]− m/z 672 in negative mode by LC-MS. Phe (F) is known to be the most vulnerable residue to be oxidized due to its aromatic side chain.51 However, peptide IPAVFK at [M − H]− m/z 672 was relatively stable and not easily and rapidly oxidized. There were no oxidation products significant enough to be quantified for peptide IPAVFK ([M + H]+ m/z 674), as compared to the peptide LIVTQTMK ([M + H]+ m/z 934) and the peptide ALPMHIR ([M + H]+ m/z 838), in positive mode detection by MS. Total ion chromatograms in Figure 11 were acquired from LCESI-MS in negative mode detection through oxidation day 0 to day 7. Two sets of samples were monitored, one is fractionated βLg peptide IPAVFK and the other is fractionated peptide IPAVFK with 1000 μg/g sanguiin H-6. When the peak integration was done by using the total ion chromatogram, several different analytes eluted simultaneously. The amount of peptide IPAVFK did not change considerably in the samples with and without sanguiin H-6 on day 0. The same occurred also on day 7. Fewer peaks could be detected in the sample with sanguiin H-6 than without both on day 0 and day 7. The changes of peak areas of interaction products were not significant enough to be observed in the total ion chromatograms. In negative mode, there were fewer peaks in the sample with sanguiin H-6 than that without. This may be because the scavenging activity of sanguiin H-6. Sanguiin H-6 interacted with other nontarget peptides and stabilized the reaction system. On day 7, unoxidized form of peptide IPAVFK ([M − H]− m/z 672) was still existing but slightly decreased as compared to day 0.

Figure 10. Changes in the amounts of peptide ALPMHIR and its oxidation products in the samples with and without 1000 μg/g sanguiin H-6 in positive mode detection in (a) ALPMHIR ([M + H]+ m/z 838), (b) [ALPMHIR + O] ([M + H]+ m/z 854), and (c) [ALPMHIR + 2O] ([M + H]+ m/z 870).

The general trends of amounts for the peptide ALPMHIR ([M + H]+ m/z 838) and its oxidation products ([M + H]+ m/z 854 and [M + H]+ m/z 870) were very analogous. They all decreased from day 0 to day 1 and increased through day 1 to day 7. The difference between the samples with and without sanguiin H-6 was that the changes of peptide and the amounts of its oxidation products were more stable without sanguiin H-6 than those with sanguiin H-6, thus contradicting the findings with peptide LIVTQTMK. Moreover, the rate of decline for the ion m/z 870 was faster than for the ions of m/z 838 and m/z 854 from day 0 to day 7 (Figure 10). On day 0, both monoxidized and dioxidized forms of peptide ALPMHIR (m/z 854 and m/z 870) were detected. The amount of unoxidized peptide ALPMHIR (m/z 838) was higher in the samples with sanguiin H-6 than without sanguiin H-6 because of the antioxidant or scavenging activity of sanguiin H-6 (Figure 10a). For [ALPMHIR + O], the amount in the sample with sanguiin H-6 was similar to that without sanguiin H-6 (Figure 10b). The reason might be that the oxidation of peptide ALPMHIR took place even more rapidly than the fast radical scavenging activity of sanguiin H-6. Therefore, the radical scavenging activity of sanguiin H-6 was not significant on day 0 for the very first oxidation step of peptide ALPMHIR. For the 5035

dx.doi.org/10.1021/jf501190x | J. Agric. Food Chem. 2014, 62, 5028−5037

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Figure 11. TIC of peptide IPAVFK with and without sanguiin H-6 in negative mode. (a) TIC of peptide IPAVFK without sanguiin H-6 on day 0. (b) TIC of peptide IPAVFK with 1000 μg/g of sanguiin H-6 on day 0. (c) TIC of peptide IPAVFK without sanguiin H-6 on day 7. (d) TIC of peptide IPAVFK with 1000 μg/g of sanguiin H-6 on day 7.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: Marina.Heinonen@helsinki.fi. Tel.: +358-2-941-58224. Notes

The authors declare no competing financial interest.

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