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. Author manuscript; available in PMC: 2013 Sep 26.
Published in final edited form as: J Agric Food Chem. 2012 Sep 11;60(38):9727–9736. doi: 10.1021/jf3026277

Mass Spectrometric Evidence of Malonaldehyde and 4-Hydroxynonenal Adductions to Radical-Scavenging Soy Peptides

Jing Zhao †,§, Jing Chen ‡,§, Haining Zhu ‡, Youling L Xiong †,*
PMCID: PMC3580276  NIHMSID: NIHMS407486  PMID: 22946674

Abstract

Antioxidative peptides in food systems are potential targets of lipid oxidation-generated reactive aldehydes, such as malonaldehyde (MDA) and 4-hydroxynonenal (HNE). In this study, covalent modifications on radical-scavenging peptides prepared from soy protein hydrolysate by MDA and HNE were characterized by liquid chromatography–electrospray ionization-mass spectrometry (LC-ESI-MS/MS). MS/MS analyses detected the formation of Schiff base type adducts of MDA on the side chain groups of lysine, histidine, arginine, glutamine, and asparagine residues as well as the N-termini of peptides. MDA also formed a fluorescent product with lysine residues. HNE adducted on lysine residues through Schiff base formation and on histidine, arginine, glutamine, and asparagine residues mainly through Michael addition. In spite of the extensive MDA modification, peptide cross-linking by this potential mechanism was undetectable.

Keywords: radical-scavenging activity, peptides, malonaldehyde (MDA), 4-hydroxynonenal (HNE), mass spectrometry

INTRODUCTION

Antioxidant peptides have been reported to behave as sacrificial radical scavengers and metal chelators in food systems thus prevent other food components from being attacked. Our preliminary study showed that during the inactivation of hydroxyl radicals, some amino acid residues in soy protein hydrolysate, such as methionine, histidine and lysine, were modified (unpublished data). The covalent modifications of amino acid residues during protein oxidation can be induced either directly by reactive oxygen species or indirectly by reaction with secondary products of lipid oxidation, e.g., malonaldehyde (MDA) and 4-hydroxynonenal (HNE).1 MDA and HNE, the two major reactive aldehyde species formed during polyunsaturated fatty acid peroxidation, are well known for their detrimental effects on biomolecules (proteins, DNA, phopholipids, etc.) and involvement in human diseases, e.g., neurodegenation.2–4 These aldehydes are more stable than free radicals, thus, can diffuse to a target far from the site of their formation,3 resulting in different modification sites on peptides from those attacked by free radicals.

Lipid oxidation can exert various detrimental effects on food products. One of the most notorious effects is off-flavors generated from oxidation of unsaturated fatty acids. It also changes the type and concentration of molecular species present in a food,5 thus influence the quality attributes of foods, such as taste, texture, shelf-life, appearance, and nutrition profile.6 For example, the adduction of HNE on myoglobin induces the discoloration of meat which leads to a marked loss of products’ commercial value.7 In order to prevent the deleterious effects of lipid oxidation in foods, antioxidative peptides have been applied, for example in meat products and in emulsions where tremendous lipid oxidation can occur.8,9 Because by virtue of inactivating reactive oxygen species when used to protect other food components, antioxidative peptides are sacrificed, it is of interest to understand the fate of these peptides following their reaction with reactive aldehydes generated from lipid oxidation.

The adduction of MDA and HNE on amino acids has been widely observed. MDA, a strong alkylating agent with two reactive aldehyde groups positioned on the opposing ends of the molecule, forms a Schiff base complex predominantly with primary amino groups such as ε-amines of lysine residues and the N-termini of the peptide chains.10 MDA has also been reported to react with histidine in bovine serum albumin.11 In addition, MDA adduction can generate fluorescent compounds such as the dihydropyridine (DHP)-type adducts.

HNE is another highly reactive bifunctional aldehyde. The conjugation of the double bond with the aldehyde group and the electron-withdrawing 4-hydroxyl group in HNE make the central carbon (C-3) extremely susceptible to nucleophilic attack by free amines. Thus, HNE forms Michael adducts with nucleophilic sites in proteins, such as histidine imidazole moieties, cysteine sulfhydryls, and ε-amino groups of lysine residues.12 These Michael adducts are stabilized in the form of cyclic hemiacetals. Besides forming Michael adducts, the aldehyde group in HNE is able to form Schiff bases with the N-termini of peptide chains and ε-amino groups of lysine residues in proteins.13 Because of the bifunctional nature of both MDA and HNE, they all have the potential to cross-link proteins.14

A previous study demonstrated an MDA concentration-dependent increase in the protein carbonyl content and decreases in the free sulfhydryl, disulfide, free amine, and lysine content of soy protein after reacting with MDA (0–100 mM, 25 °C, 24 h), indicating the adduction of MDA on cysteine, lysine, and the N-termini of soy protein polypeptide chains.15 Uchida and Stadtman16 suggested that the primary target of HNE on different proteins can vary, e.g., lysine in low density lipoproteins and glucose-6-phosphate dehydrogenase, and histidine in insulin.

In the present study, mass spectrometry was applied to identify the MDA and HNE modification sites on chromatographically separated radical-scavenging soy peptides. Such an analytical approach has the merit of high degree of confidence in the assignments and potential to identify the precise sites within the peptide chains that are modified or cross-linked.17 To our knowledge, this was the first study in which mass spectrometry was used to reveal the modifications of radical-scavenging peptides by secondary lipid oxidation products.

MATERIALS AND METHODS

Materials

Soybeans were purchased from Bonnie Buhs Co. (Gibson, IN). Soy protein isolate, protein hydrolysates, and peptide fractions were prepared in the lab. HNE was obtained from Cayman Chemical Co. (Ann Arbor, MI), and 1,1,3,3-tetraethoxyproane (MDA precursor) was from Sigma Chemical Co. (St. Louis, MO). Alcalase® 2.4 L FG (2.4 AU/g) was obtained from Novo Nordisk (Bagsvard, Denmark). All other chemicals, all reagent grade, were purchased from Fisher Scientific (Indianapolis, IN) or Sigma Chemical Co.

Preparation of Soy Protein Hydrolysate (SPH)

Soy protein was isolated from defatted soy flour using isoelectric precipitation as described by Jiang et al.18 An SPH with a degree of hydrolysis of 5%, previously established to be strongly antiradical,1 was prepared with AlcalaseR at pH 8, 50 °C, and 1/100 enzyme/protein ratio. Degree of hydrolysis was controlled by the pH-stat method.19 Prior to hydrolysis, the protein substrate was heat-treated at 90 °C for 5 min to improve enzyme accessibility. When a degree of hydrolysis of 5% was reached, the hydrolysate was heated at 80 °C for 15 min to inactivate Alcalase®, adjusted back to pH 7, freeze-dried, and stored in a freezer until use.

Peptide Fractionation

SPH was subjected to peptide fractionation using low-pressure gel filtration chromatography with a 2.6 cm (dia.) × 70 cm (length) Sephadex G-25 fine column (Pharmacia XK 26/70, Piscataway, NJ) according to Ma et al. 20 SPH (1% protein) dissolved in deionized water was filtered through a 0.22 Qm membrane, and an aliquot of 6 mL was loaded to the column for separation. Peptides were eluted with filtered deionized water at 4 °C and a flow rate of 0.9 mL/min. The eluents were collected with an automatic fraction collector with a collection time of 6 min for each collection tube. Protein concentration in each tube was measured with a UV/Vis spectrometer at 280 nm. The corresponding peptide fractions from 20 chromatographic runs were pooled, lyophilized, and kept in a freezer for further analysis. The average molecular weight of each individual peptide fraction was estimated from a calibration curve generated from the elution volume of the following standards: cytochrome C (12327 Da), aprotinin (6512 Da), bacitracin (1423 Da), and tetrapeptide GGYR (452 Da) according to Ma et al.20

Measurement of Radical-Scavenging Capacity

Freeze-dried peptide fractions were dissolved in aqueous solutions. Protein concentration was measured with bicinchoninic acid (BCA) using a Pierce BCA protein assay kit purchased from Thermal Fisher Scientific Inc. (Rockford, IL). Samples adjusted to the same protein concentration levels were subjected to the radical-scavenging activity assay using 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) radicals (ABTS+•) according to Pellegrini et al. 21 The absorbance reading (734 nm) was taken after 10 min and converted to “Trolox equivalent antioxidant capacity” (TEAC, mM) based on a standard curve generated with Trolox.

Analysis of Amino Acid Composition

Acid hydrolysis of peptide samples prepared from the gel filtration fractionation was performed with 6 M HCl at 110 °C for 24 h in glass tubes purged with nitrogen gas before seal. The amino acid composition was analyzed by reverse-phase high-pressure liquid chromatography with a 3.9 mm × 300 mm Nova-Pak C18 column (Waters Co., Millford, MA). Norleucine was added as the internal standard. Mobile phase A was prepared by mixing reagent 1 [1.9% (w/v) sodium acetate trihydrate, 0.05% (v/v) triethylamine, and 0.2 ppm ethylenediaminetetraacetic acid (EDTA) titrated to pH 6.40 with glacial acetic acid] and acetonitrile at a 47:3 (v/v) ratio. Mobile phase B was prepared by mixing acetonitrile, deionized water, and 1000 ppm EDTA at a 3000:2000:1 (v/v/v) ratio. Both eluent solutions were degassed before use.

Reaction with MDA

MDA was prepared by the hydrolysis of bis 1,1,3,3-tetraethoxypropane (23 mg) in 0.1 M HCl (10 mL) at 40 °C for 40 min. The pH of MDA solution was adjusted to 7.2 with 6 M NaOH. MDA concentration was measured at 245 nm in 1% H2SO4 and calculated using the molar extinction coefficient of 13700 M−1cm−1.22 Peptide samples dissolved in 10 mM phosphate buffer (pH 7.2) and MDA were mixed at final concentrations of 1 mg/mL and 5 mM, respectively, and incubated at 37 °C for 24 h.

Reaction with HNE

HNE solution was prepared fresh by dilution to 4 mM with a 10 mM phosphate buffer (pH 7.2). Peptide samples (2 mg/mL) in the same buffer were mixed with the prepared HNE at a 1:1 ratio and incubated at 37 °C for 6 h. One half of the reacted sample was subjected to mass spectrometry (non-reduced) and the other half was treated with sodium borohydride (NaBH4) at a concentration of 1.0 M to stabilize the reversible HNE–lysine adducts in the reduced form before mass spectrometry.23

Liquid Chromatography-Electrospray Ionization-Tandem Mass Spectrometry (LC-ESI-MS/MS)

LC-MS/MS analysis was performed using an LTQ-Orbitrap mass spectrometer (Thermo Fisher Scientific, Waltham, MA) coupled with an Eksigent Nanoflex cHiPLC™ system (Eksigent, Dublin, CA) through a nano-electrospray ionization source. The peptide samples were separated using a reversed phase cHiPLC column (75 µm × 150 mm) at a flow rate of 300 nL/min. Mobile phase A was deionized water with 0.1% (v/v) formic acid while B was acetonitrile with 0.1% (v/v) formic acid. A 40 min gradient condition was applied: initial 3% mobile phase B was increased linearly to 50% B in 24 minutes and further to 85% B for 8 minutes before it was decreased to 3% B for re-equilibration. The eluted peptides were analyzed using data-dependent acquisition: all peptides eluted from the cHiPLC column at a particular elution time were analyzed by mass spectrometry using Orbitrap (m/z 100–1,600) with a resolution of 60,000. From each peptide MS spectrum, seven most abundant peptides were subjected to collision induced dissociation (CID) and MS/MS analysis in LTQ linear trap.

Identification of Peptides and MDA/HNE Adduction Sites

The LC-MS/MS data were submitted to a local Mascot server for an MS/MS identification search against a customized database using Proteome Discoverer software. Peptide tolerance and MS/MS tolerance were set as 10 ppm and 0.8 Da, respectively. The database encompasses the sequences of five polypeptides from soy glycinin (G1, G2, G3, G4, and G) and three subunits of soy β-conglycinin (α, α', and β chains) obtained from UniProtKB/Swiss-Prot (www.expasy.org/sprot). A decoy database was built and searched. Filter settings that determine false discovery rates (FDR) were used to distribute the confidence indicators for the peptide matches. Peptide matches that passed the filter associated with the strict FDR (with target setting of 0.01) were assigned as high confidence. Mass additions of 54 Da and 134 Da were searched to identify MDA Schiff base adduction and fluorescent DHP-lysine adduct, respectively. For HNE Michael adducts, the mass increments were 156 Da in non-reduced samples and 158 Da for the reduced adduction products. In the case of cysteine–HNE conjugate, a neutral loss of HS–HNE moiety was examined according to Wu.24 HNE Schiff base formation was searched with mass addition of 138 Da and 140 Da in non-reduced and reduced samples, respectively. A reported stable pyrrole type HNE–lysine adduct, which has a mass addition of 120 Da, was examined in non-reduced samples.

Identification of MDA-induced Cross-linking

MDA-modified peptides were analyzed in an attempt to identify MDA-induced cross-linking. Reaction of peptides and MDA was done in the same way as described above. After 24 h incubation, the reaction was terminated by increasing the pH to 9. Samples were reduced with NaBH4 before being subjected to acid hydrolysis in 6 M HCl at 110 °C for 24 h. The hydrolyzed samples were freeze-dried and re-dissolved in 10 mM phosphate buffer (pH 7.2) for LC-MS/MS analysis.

Statistical Analysis

Radical-scavenging activity tests were repeated three times as independent trials (replicates). The data were subjected to the analysis of variance using the general linear model’s procedure of the Statistix software 9.0 (Analytical Software, Tallahassee, FL). Significant (P < 0.05) differences between means were identified by least significant difference all-pairwise multiple comparisons. The LC-MS/MS experiments were repeated twice.

RESULTS AND DISCUSSION

Radical-Scavenging Capacity

Previous studies have shown that the size of peptides plays a role in their antioxidant activity.25 Therefore, size-exclusion gel filtration chromatography was applied to separate peptides in SPH (Figure 1), which were subsequently grouped into five molecular weight fractions. Antioxidant activity of the peptide fractions was estimated using the ABTS+• scavenging test (Figure 1). The two large molecular weight fractions (I and II, at 1719 and 746 Da, respectively) exhibited lower radical-scavenging capability comparing to SPH, whereas the scavenging activity was improved in small molecular weight fractions (III–V). Peptide fraction IV displayed a significantly higher (P < 0.05) antiradical potential than any other fractions and was two-fold more potent than SPH. Hence, it was selected for subsequent oxidation analyses. The estimated mean molecular weight of fraction IV was 401 Da, corresponding to about 4 amino acids. However, the fraction could contain peptides with as many as 6 amino acid residues or as few as 3 amino acids when heading (461 Da) and tailing (369 Da) portions of the fraction were considered (Figure 1). The result agreed with literature reports that typical antioxidative peptides are comprised of 2–9 amino acids.26

Figure 1.

Figure 1

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Radical-scavenging activity (lower panel) of soy peptide fractions prepared with gel-filtration chromatography (upper panel). The result is expressed as Trolox equivalent antioxidant capacity (TEAC). The labels above the peptide fractions (upper panel) indicate the estimated mean molecular weight of each fraction.

Amino Acid Composition

The composition of amino acids in the above separated peptide fractions is presented in Table 1. The two fractions that exhibited the best radical-scavenging activity (IV and V) contained significantly higher (P < 0.05) amounts of histidine, tyrosine, and phenylalanine than SPH and other fractions. Histidine content in fractions IV and V was 3.72 and 3.91 g/100 g protein, respectively, compared with 2.08–2.92 g/100 g protein in SPH and other fractions. The two aromatic amino acids tyrosine (17.14 g/100 g protein) and phenylalanine (18.31 g/100 g protein) were 2–6 fold more abundant in fraction IV, which exhibited the highest radical-scavenging activity of all, than in SPH and fractions I–III.

Table 1.

Amino Acid Composition of Soy Protein Hydrolysate (SPH) And Its Fractions (I-V) Separated By Sephadex Gel Filtration. a

amino acid SPH
(g/100g protein)		 I
(g/100g protein)		 II
(g/100g protein)		 III
(g/100g protein)		 IV
(g/100g protein)		 V
(g/100g protein)
Asp+Asn 12.66±0.08 a 10.86±1.16 b 12.42±0.59 a 10.79±0.55 b 7.02±0.25 c 5.84±0.18 d
Glu+Gln 19.94±0.39 bc 28.11±5.27 a 23.07±0.58 b 17.99±0.66 c 10.48±0.43 e 14.29±0.38 d
Ser 4.78±0.15 a 3.97±0.64 b 3.79±0.16 b 5.24±0.16 a 4.11±0.70 b 2.66±0.08 c
Gly 3.50±0.05 b 3.16±0.28 c 3.44±0.10 b 3.63±0.07 b 3.60±0.14 b 4.03±0.08 a
His 2.63±0.11 b 2.92±0.42 b 2.08±0.13 c 2.16±0.16 c 3.72±0.42 a 3.91±0.42 a
Arg 8.60±0.10 bc 10.39±1.24 a 8.12±0.08 c 7.78±0.08 c 6.84±0.73 d 9.16±0.21 b
Thr 3.36±0.07 b 2.68±0.45 c 3.64±0.07 ab 3.98±0.13 a 2.17±0.35 d 2.45±0.05 cd
Ala 3.72±0.04 b 2.54±0.81 c 3.57±0.10 b 5.41±0.10 a 3.60±0.16 b 2.66±0.09 c
Pro 5.23±0.03 c 7.23±1.35 a 6.28±0.08 b 4.01±0.08 d 2.38±0.04 e 5.05±0.09 c
Tyr 3.84±0.06 c 2.25±1.00 d 2.34±0.05 d 3.48±0.07 c 17.14±0.24 a 13.44±0.06 b
Val 4.81±0.07 b 3.26±0.99 d 5.46±0.06 a 5.50±0.21 a 3.33±0.15 d 4.07±0.14 c
Met 1.21±0.06 a 1.11±0.04 a 0.92±0.06 b 0.99±0.09 b 0.74±0.09 c 0.75±0.04 c
Ile 5.02±0.06 b 3.99±0.66 c 6.02±0.04 a 5.76±0.12 a 3.51±0.34 d 4.59±0.10 b
Leu 8.06±0.10 c 5.80±1.44 d 7.54±0.02 c 10.43±0.18 b 8.40±0.60 c 12.78±0.10 a
Phe 5.61±0.05 c 3.96±1.07 d 3.90±0.03 d 6.24±0.07 bc 18.31±1.31 a 6.95±0.09 b
Lys 7.04±0.07 bc 7.75±0.54 a 7.42±0.09 ab 6.62±0.04 c 4.64±0.42 d 7.37±0.35 ab
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a

Means within the same row (same amino acid) sharing no common letter (a–e) differ significantly (P < 0.05).

The relative abundance of histidine, tyrosine, and phenylalanine residues in the radical-scavenging peptide fraction (IV) supported previous findings that phenylalanine, tyrosine and histidine were the main amino acids responsible for peptide antioxidant activity.27–29 Interestingly, the total amounts of tyrosine and phenylalanine, which were 9.45, 6.21, 6.24, 9.72, 35.46, and 20.38 g/100 g protein in SPH and fractions I–V, respectively (Table 1), corresponded well (linear R2 = 0.84; quadratic R2 = 0.92; P < 0.05) with their radical-scavenging activity (Figure 1). Aromatic amino acids are believed to contribute to the antioxidant activity and radical-scavenging potential of several protein sources, e.g., hydrolyzed potato protein,27 egg yolk,30 and pea seed protein hydrolysate.31

Peptide Identification

The selected radical-scavenging peptide fraction (IV) was subjected to LC-ESI-MS/MS to identify the peptide sequences. More than 100 peptides derived from glycinin (G1–4, G) and β-conglycinin (α', α, and β) were identified from Mascot search with high confidence. The peptides sequenced had a chain length of 5–14 amino acid residues with the majority of them consisting of 5–9 residues (results not shown). These peptides covered about 17% of the α and β subunits and 21% of α' subunit of β-conglycinin, as well as 29% of each subunits of glycinin except glycinin G3 (16%). Lys–His–Glu–Trp–Gln–His–Lys and Leu–Arg–Asp–Tyr–Arg–Ile from β-conglycinin and Phe–Leu–Lys–Tyr–Gln from glycinin have been detected to be highly abundant. The average molecular weight of peptides identified by MS was larger than that estimated by the gel filtration (up to about 6 amino acid residues) probably due to the technical limitation of Sephadex gel filtration as an accurate tool to estimate the molecular weight of macromolecules.32

Identification of MDA/HNE Adduction Sites

MDA adduction

Reaction of MDA with amino acid residues in peptides and proteins mainly took place in the form of Schiff base formation at the nucleophilic amine groups, such as lysine, histidine, arginine, and the amino termini.23 In addition, MDA is capable of cross-linking proteins and producing fluorescent adducts such as DHP-lysine.33,34 The predicted structures of MDA adduction products are summarized in Table 2.

Table 2.

Predicted Structures of Detected Aldehyde Adducts to Amino Acid Residues in Soy Peptides. MDA: Malonaldehyde; HNE: 4-Hydroxynonenal. ND: Not Detected.

graphic file with name nihms407486t1.jpg
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Mascot search with a mass increment of 54 was applied to identify MDA Schiff base formation with the side chain groups of lysine, histidine, arginine, glutamine, and asparagine residues as well as the N-termini of the peptide chains. Although the reactivity of glutamine and asparagine with aldehydes is expected to be low due to the reduced nucleophilicity of the amino group in an amide where the carbonyl group is present at the alpha position, both amino acid residues were included in the search. A total of 53 peptides were identified to react with MDA through Schiff base adduction. More than half of the modifications took place on the N-termini of the peptides. This could be a result of smaller steric hindrance at the terminus than within the sequence and the abundance of N-terminal residues as a result of protein hydrolysis (peptide bond cleavage). MDA Schiff base formation was detected at the side chains of lysine, histidine, arginine, asparagine, and glutamine. Surprisingly, MDA adduction on arginine residues occurred in a large number of peptides.

The MS/MS results for some of these MDA adducts are shown in Figures 2–4. Both single and double adductions were observed on peptides with two or more nucleophilic sites. As an example, Figure 2 shows the MDA simultaneous adductions on histidine and lysine (A) and a single adduction on glutamine (B) in peptide Lys–His–Glu–Trp–Gln–His–Lys, which was one of the prominent peptides identified in the strongly antiradical fraction IV. Figure 3 is an example of MDA adduction on the arginine side chain amino group in Trp–His–Arg–Lys–Glu–Glu. Figure 4 shows MDA Schiff base formation on asparagine side chain (A) and on the N-terminal amino group serine (B) in Ser–Asn–Leu–Asn–Phe–Phe.

Figure 2.

Figure 2

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Malonaldehyde Schiff base adduction (*, +54 Da, **, +108 Da) on lysine (A), histidine (A), and glutamine (B) side chain groups in Lys–His–Glu–Trp–Gln–His–Lys.

Figure 4.

Figure 4

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Malonaldehyde Schiff base adduction (*, +54 Da) on asparagine side chain group (A) and the N-terminus serine (B) in Ser–Asn–Leu–Asn–Phe–Phe.

Figure 3.

Figure 3

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Malonaldehyde Schiff base adduction (*, +54 Da) on arginine side chain group in Trp–His–Arg–Lys–Glu–Glu.

In addition to the Schiff base type adducts, a fluorescent DHP type adduct on lysine residues was detected in Lys–His–Glu–Trp–Gln–His–Lys, Lys–Tyr–Glu–Gly–Asn–Trp–Gly–Pro–Leu, Phe–Lys–Asn–Gln–Tyr–Gly–His–Val–Arg, Gly–Arg–Lys–Gln–Gly–Gln–His–Gln–Gln, and Gln–Lys–Gln–Lys–Gln–Glu–Glu–Glu. The MS/MS evidence of DHP–lysine formation in Lys–His–Glu–Trp–Gln–His–Lys is shown in Figure 5.

Figure 5.

Figure 5

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Dihydropyridine (DHP) type fluorescent malonaldehyde adduct (*, +134 Da) on lysine residue in Lys–His–Glu–Trp–Gln–His–Lys.

HNE adduction

HNE is a readily diffusible and selective electrophile that is a key mediator of oxidative stress.3 The Michael adducts of HNE on amino acid residues have a 156 Da (non-reduced form) or 158 Da (reduced form) mass increment. The aldehyde group in HNE is also capable of forming Schiff base adducts with nucleophilic amino groups, which has a 138 Da (non-reduced form) or 140 Da (reduced form) mass increment. The detection of HNE adductions in the present study focused on lysine, cysteine, histidine, arginine, glutamine, and asparagine.

Mascot search yielded no match of Schiff base adducts with a mass addition of 138 Da (bound HNE) in non-reduced peptides. Previous studies have shown that HNE Schiff base adduction on lysine is reversible,35,36 and the adduct is unstable during MS analysis.37 However, the pyrrole-type HNE–lysine adduct is more stable,38,39 and was detected in peptide Lys–Tyr–Glu–Gly–Asn–Trp–Gly–Pro–Leu (Figure 6A). On the other hand, in samples reduced with NaBH4, the reduced form of HNE–lysine Schiff base adduct was detected in Thr–Trp–Asn–Pro–Asn–Asn–Lys–Pro–Phe, Asn–Phe–Gly–Lys–Phe–Phe, and Lys–Tyr–Glu–Gly–Asn–Trp–Gly–Pro–Leu (Figure 7B). Michael addition products were successfully identified on histidine, arginine, glutamine, and asparagine residues but not on lysine. This observation suggested that HNE reacted with lysine residues in radical-scavenging peptides mainly through Schiff base formation. For all other amino acids, HNE modification occurred in the form of Michael addition.

Figure 6.

Figure 6

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4-hydroxynonenal (*) pyrrole type adduction (A, +120 Da) and Schiff base formation (B, +140 Da) on lysine residue detected from non-reduced and reduced Lys–Tyr–Glu–Gly–Asn–Trp–Gly–Pro–Leu, respectively.

Figure 7.

Figure 7

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4-hydroxynonenal Michael adduction (*, +156 Da) on histidine (A) and arginine (B) residues in Phe–Lys–Asn–Gln–Tyr–Gly–His–Val–Arg from non-reduced peptides.

In non-reduced samples, histidine residues were found to be the major targets of HNE. HNE–His Michael adducts were identified in 5 out of 22 sequenced histidine-containing peptides, namely, Phe–Lys–Asn–Gln–Tyr–Gly–His–Val–Arg (Figure 7A), Gln–Tyr–Gly–His–Val–Arg, Phe–Val–Pro–His–Tyr–Asn–Lys–Asn, His–Phe–Leu–Ala–Gln, and Thr–Trp–Asn–Ser–Gln–His–Pro–Glu. In addition, HNE Michael addition was detected in Phe–Lys–Asn–Gln–Tyr–Gly–His–Val–Arg on arginine side chain group (from both non-reduced and reduced samples) (Figure 7B) and on glutamine side chain groups in Thr–Trp–Asn–Ser–Gln–His–Pro–Glu (from non-reduced samples), Phe–Lys–Asn–Gln–Tyr–Gly–His–Val–Arg (from reduced samples) (Figure 8A), and Lys-His-Glu-Trp-Gln-His-Lys (from reduced samples). Previously, HNE Michael adduction on arginine residue has been reported in cytochrome c by Isom et al.39 However, no HNE adduction on glutamine residue has been reported so far. Interestingly, the modified arginine and glutamine residues were all either beside a histidine residue or were one amino acid away from a histidine residue in the sequence. We assume that the detected HNE adduction on arginine and glutamine residues might be a result of HNE transfer between amino acid side chain groups.

Figure 8.

Figure 8

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4-hydroxynonenal Michael adduction (*, +158 Da) on glutamine residue in Phe–Lys–Asn–Gln–Tyr–Gly–His–Val–Arg (A) and asparagine residue in Ser–Asn–Leu–Asn–Phe–Phe (B) from peptides reduced with NaBH4.

In the reduced samples, HNE adduction on asparagine was found in Ser–Asn–Leu–Asn–Phe–Phe (Figure 8B), Phe–Lys–Asn–Gln–Tyr–Gly–His–Val–Arg, Gly–Val–Ala–Trp–Trp–Met–Tyr–Asn–Asn–Glu–Asp–Thr–Pro–Val–Val–Ala–Val, and Lys–Tyr–Glu–Gly–Asn–Trp–Gly–Pro–Leu, which was not identified in non-reduced samples. The results suggested that HNE–asparagine adducts were not stable during MS analysis and the adduction reaction could be reversible.

It has been reported that HNE also reacts with cysteine residues, and sulfhydryl groups were the primary nucleophilic targets of HNE.40 In our case, only one cysteine-containing peptide [Pro–Gln–Cys–Lys–Gly–Lys–Asp from glycinin G1 (A1a, Bx)] was detected from the radical-scavenging peptide fraction (IV) selected for mass spectrometry and HNE adduction study. Besides, according to the pKa of cysteine side chain group (which is around 8.3), at the HNE reaction pH (pH 7.2), less than 10% of sulfhydryl groups would be in the deprotonated active form. Therefore, even the reactivity of cysteine at neutral pH is the greatest among the nucleophilic amino acids (Cys, His, Lys, Arg),41 it was not surprising that no HNE adduction on cysteine residues had been identified in our study.

Identification of MDA-Induced Cross-Linking

Because of the technical difficulty of performing Mascot search for cross-linking, an indirect approach was taken to verify MDA-induced peptide cross-linking. Since Schiff base is unstable in acidic conditions, MDA modified peptides were stabilized in their reduced form by reacting with NaBH4. After acid hydrolysis, which released individual amino acids, the cross-linked complexes, for example, lysine–MDA–lysine, were searched in the MS spectrum. Although MDA single adduction on individual amino acids (e.g., lysine and histidine) was successfully detected, no cross-linked compounds were found, demonstrating the lack of cross-linking by MDA in these radical-scavenging peptides. This seems understandable because peptides of 5–14 amino acid residues should be quite flexible, mobile and elusive in solution when compared with large proteins, and the conceivable strong electrostatic repulsions could further prevent peptides from getting close enough so as to form complexes.

In conclusion, a strongly antiradical fraction from soy protein hydrolysate contained peptides (5–14 amino acid residues) that were readily reactive with MDA and HNE through Schiff base formation or Michael addition. Besides the modification on lysine, histidine, arginine, and the N-terminal amino acid residues, MDA also adducted on glutamine and asparagine in a number of soy peptides. The modification of lysine by HNE was primarily through Schiff base formation, but the modification of other nucleophilic amino acid side chain groups by HNE was via Michael addition. Nonetheless, MDA was unable to generate cross-linked products among antiradical small soy peptides probably due to electrostatic repulsions between peptides, steric hindrances, and their high mobility in the aqueous solution. The radical-scavenging activity, coupled with the reactivity with potent secondary products from lipid oxidation by many of the peptides, may explain why enzymatic hydrolysis improves the antioxidant activity of soy protein as demonstrated in previous studies.

ACKNOWLEDGMENTS

This research was supported by NRI/CSREES/USDA (Grant 2008-35503-18790). We acknowledge the proteomics core that is partially supported by grants from the National Center for Research Resources (P20RR020171-09) and the National Institute of General Medical Sciences (P20GM103486-09) from the National Institutes of Health. The Orbitrap mass spectrometer was acquired by a High-End Instrumentation grant S10RR029127 (to H.Z.). Approved for publication as journal article 12-07-058 by the Director of the Kentucky Agricultural Experiment Station.

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