Detection of 4-hydroxy-2-nonenal adducts of turkey and chicken myoglobins using mass spectrometry

Food Chemistry 122 (2010) 836–840

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Short communication

Detection of 4-hydroxy-2-nonenal adducts of turkey and chicken myoglobins using mass spectrometry B.M. Naveena a,*, C. Faustman b, N. Tatiyaborworntham b, S. Yin b, R. Ramanathan b, R.A. Mancini b a b

National Research Centre on Meat, Chengicherla, Hyderabad, Andhra Pradesh 500039, India Department of Animal Science, University of Connecticut, Storrs, CT 06269, USA

a r t i c l e

i n f o

Article history: Received 27 September 2009 Received in revised form 3 January 2010 Accepted 23 February 2010

Keywords: Mass spectrometry 4-Hydroxy-2-nonenal Myoglobin Lipid oxidation

a b s t r a c t 4-Hydroxy-2-nonenal (HNE), an unsaturated aldehyde generated by peroxidation of polyunsaturated fatty acids, is highly reactive and destabilizes myoglobin (Mb) redox state, affecting meat colour. Our objective was to characterise the adduction of HNE to turkey and chicken Mbs using tandem mass spectrometry (MS/MS). Turkey and chicken oxymyoglobins (OxyMbs) were incubated with HNE at 25 °C, pH 5.8 or 7.4. MetMb formation was greater in the presence of HNE than controls (p < 0.05). Electrospray ionisation-Q-TOF mass spectrometry of HNE-reacted Mbs revealed covalent adduction of HNE to both turkey and chicken Mbs via Michael addition. LC–ESI-MS/MS of chicken Mb reacted with HNE identified covalent adduction of histidine (His) residues 64 and 93 at pH 7.4, whereas at pH 5.8 only His 64 was adducted. These results suggest that HNE accelerates chicken OxyMb oxidation in vitro by covalent modification at histidine residues. Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction Meat colour is attributed to myoglobin (Mb), a haem-containing sarcoplasmic protein (Livingston, Lamar, & Brown, 1983) that can exist in any of three forms dictated by the redox state and nature of the sixth ligand species of the haem iron. Oxidation of ferrous oxymyoglobin (OxyMb) to ferric brown metmyoglobin (MetMb) is associated with meat discolouration and results in lost value due to price reductions (Smith, Belk, Sofos, Tatum, & Williams, 2000). Redox stability of Mb is impaired by many factors, including lipid oxidation products (Faustman & Cassens, 1990). a,b-Unsaturated aldehydes are a specific class of lipid oxidation products that can readily diffuse into the cytoplasm, where they react with biomolecules (Esterbauer, Schaur, & Zollner, 1991). It has been suggested that covalent binding of a,b-unsaturated aldehydes to OxyMb may alter the protein’s tertiary structure and increase susceptibility to oxidation (Alderton, Faustman, Liebler, & Hill, 2003; Suman, Faustman, Stamer, & Liebler, 2007). Among a,b-unsaturated aldehydes, 4-hydroxy-2-nonenal (HNE) is commonly identified and generated through the b-cleavage of hydroperoxides derived from n-6 polyunsaturated fatty acids (Esterbauer et al., 1991). HNE was reported to accelerate oxidation of tuna (Lee, Joo, Alderton, Hill, & Faustman, 2003), equine (Faustman, Liebler, * Corresponding author. Tel.: +91 40 27204258; fax: +91 40 27201672. E-mail address: [email protected] (B.M. Naveena). 0308-8146/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2010.02.062

McClure, & Sun, 1999), bovine (Alderton et al., 2003) and porcine (Suman et al., 2007) Mbs. Faustman et al. (1999) noted that formation of Mb-aldehyde adducts occurred concomitantly with OxyMb oxidation. The latter finding was confirmed in beef and pork Mb reacted with HNE (Alderton et al., 2003; Suman et al., 2007). At 37 °C and pH 7.4, Faustman et al. (1999) and Alderton et al. (2003) reported mono-, di-, and tri-adducts of HNE with equine and bovine myoglobins, respectively. In contrast, Suman et al. (2007) reported only mono-adducts of HNE with porcine Mb under similar conditions. The strong electrophilic nature of the C-3 carbon makes HNE highly reactive with cellular nucleophiles; histidine residues have been commonly reported targets in proteins (Alderton et al., 2003; Faustman et al., 1999; Suman et al., 2007; Uchida & Stadtman, 1992). We recently demonstrated that turkey and chicken Mbs have identical molecular mass, and that incubating them with the unsaturated aldehydes nonenal and hexenal enhanced MetMb formation compared to controls (Naveena et al., 2009). However, the hypothesised basis for increased MetMb formation caused by covalent adduction of these aldehydes was not characterised with regards to specific amino acid locations. HNE has been utilised as a model a,b-unsaturated aldehyde for examining nucleophilic amino acid targets in other species Mbs (Alderton et al., 2003; Faustman et al., 1999; Suman et al., 2007). Hence, the present study was undertaken to characterise the reaction of turkey and chicken myoglobins with HNE and to determine the potential amino acid targets in vitro.

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2. Materials and methods

2.4. ESI-Q-TOF MS

2.1. Materials

Native and HNE-treated chicken and turkey Mb samples (20 lM) were prepared in 1:1 aqueous methanol:distiled water with 0.1% acetic acid to enhance protonation. Samples (100– 200 ll) were analysed on an electrospray ionisation-Q-TOF mass spectrometer (Model: QSTAR Elite, Applied Biosystems/MDS SCIEX, Ontario, Canada). The molecular mass of the native adducted Mbs were determined by transformation of the ESI-MS raw data into a true mass scale in the mass range between 10,000 and 60,000 daltons (Da) and obtained through the instrumentation software.

Sephacryl S-200 HR, sodium bicarbonate, sodium citrate, sodium hydrosulfite, trypsin, acetonitrile, bicinchoninic acid (BCA) protein assay kit were obtained from Sigma–Aldrich Chemical Co. (St. Louis, MO). HNE was obtained from Cayman Chem. (Ann Arbor, MI). HiTrap Diethylaminoethyl (DEAE) FF columns were obtained from Amersham Biosciences (Uppsala, Sweden). Kits for sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS–PAGE) were obtained from Bio-Rad (Hercules, CA). PD-10 columns were from Pharmacia Inc. (Piscataway, NJ). All chemicals were of reagent grade or greater purity.

2.2. Myoglobin isolation and purification Turkey Mb was isolated from cardiac muscles, whereas chicken Mb was purified from skeletal muscles of spent chicken thighs and drumsticks via ammonium sulphate precipitation and gel filtration chromatography using a Sephacryl S-200 HR gel-filtration column (Faustman & Phillips, 2001; Naveena et al., 2009). The gel-filtered Mb was further purified using DEAE-cellulose anion-exchange chromatography on HiTrap DEAE-FF columns (5 ml pre-packed columns, Amersham Biosciences). The Mb fractions collected from different batches were pooled and frozen at 80 °C.

2.3. Reaction with HNE OxyMb was prepared by hydrosulfite-mediated reduction of purified chicken and turkey Mbs (Brown & Mebine, 1969; Tang, Faustman, & Hoagland, 2004) accompanied by flushing with air. Turkey (0.05 mM) and chicken (0.015 mM) OxyMbs were each combined with 0.35 and 0.1 mM HNE, respectively, and incubated at 25 °C, pH 5.8 (50 mM sodium citrate) and 7.4 (50 mM sodium phosphate). Controls consisted of OxyMb and a volume of ethanol equivalent to that used to deliver HNE to the treatment mixture. MetMb formation was calculated according to Tang et al. (2004).

2.5. Sample preparation and LC–MS/MS analysis SDS–PAGE of native and HNE-treated chicken Mbs (after 3 h at 25 °C, pH 5.8 and 7.4) was performed under reducing conditions using a mini-gel electrophoresis unit (Model: Mini Protean II, Bio-Rad Laboratories, Inc., Richmond, CA). Each band of native and HNE-treated chicken Mb on Coomassie-stained gels was excised, destained and digested with sequencing-grade trypsin at 37 °C for 18 h. The digested peptides were extracted according to the protocol of Shevchenko, Wilm, Vorm, and Mann (1996). The peptide digests were sequenced using a high throughput LCQ ESI ion trap mass spectrometer (Thermo-Finnigan, Palo Alto, CA) equipped with a commercial nano-electrospray device. The acquired MS/MS spectra were searched against UniProt chicken protein database (2008) using the SEQUEST algorithm (Keller, Nesvizhskii, Kolker, & Aebersold, 2002). The SEQUEST output files were filtered with the use of the interface software tool INTERACT to identify peptides and proteins. 2.6. Statistical analysis Data for turkey and chicken myoglobin oxidation were analysed separately. The experimental design was a completely randomized design with repeated measures. Fixed effects included time and two treatments consisting of control and HNE and their interactions. Each experiment was replicated thrice (n = 3). Data were analysed using the mixed procedure of SAS (Version 9.1, SAS

70

60

Turkey Mb

Turkey Mb+HNE

Chicken Mb

Chicken Mb+HNE

% Met Mb

50

40

30

20

10

0 0

60

120

180

240

Minutes Fig. 1. MetMb formation (%) in turkey (0.05 mM) and chicken (0.015 mM) OxyMbs incubated with HNE (0.35 and 0.1 mM, respectively) at pH 5.8 and 25 °C. Standard error bars are indicated.

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Institute, Inc., Cary, NC) with the repeated statement to assess the repeated measures (i.e., multiple measurements taken at different time points of incubation). Least square means were generated for significant F-tests (p < 0.05) and separated using least significant differences.

ported to accelerate the oxidation of equine (Faustman et al., 1999) and bovine (Alderton et al., 2003) OxyMbs. In contrast, Suman et al. (2007) did not observe a redox destabilizing effect of HNE on porcine Mb at pH 5.6, 4 °C or 37 °C. Alderton et al. (2003) and Faustman et al. (1999) utilised ESIMS to demonstrate that HNE covalently modified bovine and equine OxyMbs, respectively, through Michael addition. Faustman et al. (1999) originally proposed that covalent modification of OxyMbs by HNE provided a potential partial explanation for the redox destabilizing effect of a,b-unsaturated aldehydes, especially HNE. Naveena et al. (2009) recently reported that only mono-adducts of HNE were observed in turkey and chicken Mbs incubated with HNE at pH 5.8 and 25 °C. When turkey (Fig. 2A) and chicken (Fig. 2B) OxyMbs were incubated with HNE at 25 °C, pH 7.4, ESIQ-TOF MS spectra revealed covalent modification with up to two molecules of HNE (di-adducts). In addition to the native (i.e., unadducted) Mb peak (17,291 Da), one larger peak (17,448 Da) and one smaller peak (17,604 Da) with mass differences of 156

3. Results and discussion The effect of HNE on MetMb formation in turkey and chicken OxyMbs at pH 5.8 and 25 °C is presented in Fig. 1. MetMb formation was greater in HNE-treated turkey and chicken Mbs relative to their controls (p < 0.05). We recently reported that turkey and chicken OxyMbs incubated with nonenal and hexenal demonstrated significant MetMb formation at 25 °C and pH 5.8 (Naveena et al., 2009). The basis for variation in MetMb formation between turkey and chicken Mbs in presence of HNE with progress in incubation in the present study is not clear. HNE was previously re-

Mb 17291

100

A

90

% Relative Abundance

80 70 60 50 40 Mb:HNE 17448

30 20

Mb:2HNE 17604

10

19836

19650

19464

19278

19092

18906

18720

18534

18348

18162

17976

17790

17604

17418

17232

17046

16860

16674

16488

16302

16116

15930

15744

15558

15372

15186

15000

0

Mass Fig. 2. ESI-Q-TOF spectra of (A) turkey OxyMb (0.05 mM) and (B) chicken OxyMb (0.015 mM) following reaction with HNE (0.35 and 0.1 mM, respectively) at 25 °C for 3 h and pH 7.4.

Mb 17292

100

B

90

70 60 50 40

Mb:HNE 17448

30 20

Mb:2HNE 17604

10

Mass Fig. 2 (continued)

19843

19676

19509

19342

19175

19008

18841

18674

18507

18340

18173

18006

17839

17672

17505

17338

17171

17004

16837

16670

16503

16336

16169

16002

15835

15668

15501

15334

15167

0 15000

% Relative abundance

80

B.M. Naveena et al. / Food Chemistry 122 (2010) 836–840

and 312 Da, respectively, were observed in both turkey and chicken Mbs. These results indicated that HNE adducts formed via Michael addition as the adduct peaks corresponded to the mass of Mb plus 156 Da, the molecular mass of HNE. The greater adduct formation observed at pH 7.4 than pH 5.6 was likely due to a greater proportion of ionizable groups associated with candidate amino acid nucleophiles being charged and therefore less reactive. To date, the amino acid sequence of turkey Mb has not been reported. Therefore, we attempted to determine the specific HNE

839

adduction sites in chicken Mb only. MS/MS analyses identified two nucleophilic His residues (His 64 and 93) for Mb:HNE reactions at pH 7.4, and one nucleophilic His residue (His 64) at pH 5.8 (Table 1). As noted above, histidine residues would be expected to carry greater positive charge at pH 5.8 than 7.4 (Barrick, Hughson, & Baldwin, 1994) and this would reduce their reactivity as with HNE. His 93 is the histidine residue proximal to the haem moiety of Mb. His 64 is the distal histidine and coordinates with oxygen or other molecules associated with the sixth ligand during the interconversion of Mb redox forms. Because His 93 and 64 lie in

Table 1 MS/MS spectral features of unadducted and HNE-adducted chicken Mb peptides. Peptide positiona

Peptide sequenceb

Modification and mass shift

pValues

Precursor m/z

pH

b and y ions identifiedc

64–74

HdGATVLTQLGK

HNE, +156.0

1.30E 3

1281.31

7.4 and 5.8

64–74

HGATVLTQLGK

Unadducted

4.70E 4

1125.31

7.4 and 5.8

80–96

GNHESELKPLAQTHdATK

HNE, +156.0

2.70E 3

2018.05

7.4

80–96

GNHESELKPLAQTHATK

Unadducted

1.00E 3

1862.05

7.4

b Ions: 294.14 (b1), 351.20 (b2), 422.27 (b3), 523.38 (b4), 622.51 (b5), 735.67 (b6), 836.78 (b7), 964.21 (b8), 1078.07 (b9), 1135.12 (b10) y Ions: 147.19 (y1), 204.24 (y2), 317.40 (y3), 445.53 (y4), 546.64 (y5), 659.80 (y6), 758.93 (y7), 860.04 (y8), 931.12 (y9), 988.17(y10) b Ions: 138.14(b1), 195.20 (b2), 266.27 (b3), 367.38 (b4), 466.51 (b5), 579.67 (b6), 680.78 (b7), 808.91 (b8), 922.07 (b9), 979.12 (b10) y Ions: 147.19 (y1), 204.24 (y2), 317.40 (y3), 445.53 (y4), 546.64 (y5), 659.80 (y6), 758.93 (y7), 860.04 (y8), 931.12 (y9), 988.17 (y10) b Ions: 58.05 (b1), 172.16 (b2), 309.30 (b3), 438.42 (b4), 525.49 (b5), 654.61 (b6), 767.77 (b7), 895.94 (b8), 993.06 (b9), 1106.22 (b10), 1177.30 (b11), 1305.43 (b12), 1406.53 (b13), 1699.67 (b14), 1770.75 (b15), 1871.86 (b16) y Ions: 147.19 (y1), 248.30 (y2), 319.38 (y3), 612.52 (y4), 713.62 (y5), 841.75 (y6), 912.83 (y7), 1025.99 (y8), 1123.11 (y9), 1251.28 (y10), 1364.44 (y11), 1493.56 (y12), 1580.64 (y13), 1709.75 (y14), 1846.89 (y15), 1961.00 (y16) b Ions: 58.05 (b1), 172.16 (b2), 309.30 (b3), 438.42 (b4), 525.49 (b5), 654.61 (b6), 767.77 (b7), 895.94 (b8), 993.06 (b9), 1106.22 (b10), 1177.30 (b11), 1305.43 (b12), 1406.53 (b13), 1543.67 (b14), 1614.75 (b15), 1715.86 (b16) y Ions: 147.19 (y1), 248.30 (y2), 319.38 (y3), 456.52 (y4), 557.62 (y5), 685.75 (y6), 756.83 (y7), 869.99 (y8), 967.11 (y9), 1095.28 (y10), 1208.44 (y11), 1337.56 (y12), 1424.64 (y13), 1553.75 (y14), 1690.89 (y15), 1805.00 (y16)

a

Amino acid positions in the chicken Mb target peptide. Amino acid sequence in the chicken Mb target peptide. Observed signals assigned as b- or y-ions are listed. Ions containing an adduct moiety are mass shifted with respect to the corresponding ions in unmodified peptides and are listed in boldface. d HNE-adducted histidine residues. b

c

A

b ions: 294 351 422 523 622 735

H#

NH2

y ions:

G 147

A 204

T

V

836

964 1078 1135

L T Q

317 445 546

659

758

L 860

G K 931

COOH

988

Precursor: 1281.31

100

Abundance

90 80 70 60 50 40 30 20 10

m/z Fig. 3. Identification of chicken myoglobin histidine residues modified via Michael addition with HNE. (A) MS/MS spectrum from the tryptic-digested peptide of HNEmodified chicken Mb. (B) MS/MS spectrum from the tryptic-digested peptide of control (native) chicken Mb. Comparison of b- and y-series ions determined that one HNE molecule modified HIS 64 via Michael addition.

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B.M. Naveena et al. / Food Chemistry 122 (2010) 836–840

B

b ions: 138 NH2

H

y ions:

195

G 988

266

A 931

367

T 860

466

V 758

579

680

808

L T Q 659

546

922

979

L

G K

445 317 204

COOH

147

Precursor: 1125.31

100

Abundance

90 80 70 60 50 40 30 10

b1+

20

m/z Fig. 3 (continued)

close proximity to the haem group, their modification by HNE would be expected to alter the haem cleft region and subsequently impact redox stability (Alderton et al., 2003). Fig. 3 presents the LC–ESI-MS/MS spectra for the tryptic peptide HGATVLTQLGK (residue numbers 64–74) for both HNE-modified (Fig. 3A) and native (Fig. 3B) chicken Mbs. The 156 Da increases in the b6, b8, and b10 ions of the HNE-modified peptide, compared with the unadducted peptide, are most prominent. All of the daughter b-series ions upstream from the adduction site (His 64) showed a consistent 156 Da shift in mass. Based on the characteristic 156 Da shift in relevant ions, it was concluded that HNE reacted with the imidazole group of His 64 via Michael addition. Previous studies have also reported a mass increment of 156 Da due to HNE adduction via Michael addition in equine (Faustman et al., 1999), bovine (Alderton et al., 2003) and porcine (Suman et al., 2007) Mbs. Alderton et al. (2003) reported a total of six bovine Mb histidine residues adducted by HNE with preferential adduction of distal His 64 and proximal His 93 under conditions similar to those used in this study. Suman et al. (2007) reported three HNE-adducted histidine residues in porcine Mb but did not observe the adduction of the proximal or distal histidines at 37 °C and pH 7.4; they demonstrated a lower susceptibility of porcine Mb to the redox destabilizing effect of HNE compared to bovine Mb. Our results suggest that the redox instability of chicken OxyMb incubated with HNE might be attributed to preferential adduction of HNE with the distal histidine (His 64) which is functionally very important for haem coordination within Mb (Prass, Berkley, & Romero-Herrera, 1983). Meat is a highly complex system and has many different sources of lipid oxidation products and also many different nucleophilic sites for reaction of a,b-unsaturated aldehydes. Hence the in vitro model comprising of avian Mbs was used to understand the fundamental basis for the redox stability of avian Mbs in the presence of HNE. Acknowledgements The financial assistance from Department of Science and Technology (DST), Government of India, BOYSCAST fellowship program (Naveena, B.M) was appreciated. We thank Dr. Mohammed Karim and Dr. David Han, UCONN Health Center, University of Connecticut, for expertise in mass spectrometry analysis.

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