Analysis of Methionine Oxidation in Myosin Isoforms in Porcine Skeletal Muscle by LC-MS/MS Analysis
Analysis of Methionine Oxidation in Myosin Isoforms in Porcine Skeletal Muscle by LC-MS/MS Analysis
Food Science of Animal Resources. 2016. Apr, 36(2): 254-261
Copyright © 2016, Korean Society for Food Science of Animal Resources
This is an open access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
  • Received : October 15, 2015
  • Accepted : March 02, 2016
  • Published : April 30, 2016
Export by style
Cited by
About the Authors
Jin-Yeon, Jeong
Institute of Agriculture & Life Science, Gyeongsang National University, Jinju 52852, Korea
Eun-Young, Jung
Division of Applied Life Science (BK21 plus), Gyeongsang National University, Jinju 52852, Korea
Tae-Chul, Jeong
Institute of Agriculture & Life Science, Gyeongsang National University, Jinju 52852, Korea
Han-Sul, Yang
Division of Applied Life Science (BK21 plus), Gyeongsang National University, Jinju 52852, Korea
Gap-Don, Kim
Department of Animal Sciences, University of Illinois at Urbana-Champaign, IL 61801, USA

The purpose of this study was to analyze oxidized methionines in the myosin isoforms of porcine longissimus thoracis , psoas major , and semimembranosus muscles by liquid chromatography (LC) and mass spectrometry (MS). A total of 836 queries matched to four myosin isoforms (myosin-1, -2, -4, and -7) were analyzed and each myosin isoform was identified by its unique peptides (7.3-13.3). Forty-four peptides were observed from all three muscles. Seventeen peptides were unique to the myosin isoform and the others were common peptides expressed in two or more myosin isoforms. Five were identified as oxidized peptides with one or two methionine sulfoxides with 16 amu of mass modification. Methionines on residues 215 (215), 438 (438), 853 (851), 856 (854), 1071 (1069), and 1106 (1104) of myosin-1 (myosin-4) were oxidized by the addition of oxygen. Myosin-2 had two oxidized methionines on residues 215 and 438. No queries matched to myosin-7 were observed as oxidized peptides. LC-MS/MS allows analysis of the oxidation of specific amino acids on specific residue sites, as well as in specific proteins in the food system.
Muscle protein oxidation occurs in a number of diseases of living tissues ( Coirault ., 2007 ; Oh-Ishi ., 2003 ) as well as in muscle foods during meat-processing and storage ( Lund ., 2011 ; Soladoye ., 2015 ). Reactive species, such as hydroxyl, superoxide, peroxyl, nitric oxide, hydrogen peroxide, and peroxynitrite, as well as transition ions, are major factors in protein oxidation ( Lund ., 2011 ; Shacter, 2000 ; Stadtman, 1993 ). These factors can lead to structural and biochemical changes of muscle proteins with modifications of the amino acid side-chains and polypeptide backbones of the muscle proteins ( Lund ., 2011 ). Consequently, protein oxidation affects the deterioration of quality in muscle foods and the consumer’s health.
To delay or prevent oxidation in muscle foods requires an understanding of the various mechanisms of protein oxidation, such as carbonylation, metal ion-catalyzed oxidation, cross-linking, hydroxylation, sulfoxidation, and nitrosylation. A number of studies have been conducted to define these mechanisms ( Armenteros ., 2009 ; Decker ., 1993 ; Garrison, 1987 ; Levine, 1984 ; Stadtman and Berlett, 1988 ; Stadtman and Levine, 2003 ). Various methods for assessing protein oxidation have also been introduced. The most commonly used method for measuring total carbonyls derived from proteins is the dinitrophenylhydrazine (DNPH) method ( Oliver ., 1987 ). Thiol oxidation, which is disulfide bond-formation in thiol-containing amino acid (cysteine) residues, can be determined using dithiobisnitropyridine (DTNP) ( Winterbourn, 1990 ). Daneshvar . (1997) introduced α-aminoadipic semialdehyde (AAS) and γ-glutamic semialdehyde (GGS) as biomarkers of oxidative lysine and arginine (or proline). Recently, proteomic and spectrometric technologies have been applied in studies on specific amino acid oxidation. Bernevic . (2011) and Ghesquiere . (2011) assessed oxidized methionine, whereas Jongberg . (2011) and Estévez . (2009) applied LC-MS to the detection of potential thiol-quinone adducts and AAS and GGS, respectively. Although the proteomic approach allows the study of oxidation of specific amino acids and proteins, few studies have been conducted on muscle foods: arginine and proline ( Stadman and Levine, 2003 ), cysteine ( Lund ., 2011 ), glutamic acid and tyrosine ( Xiong, 2000 ).
In the present study, the myosin protein of porcine skeletal muscle was used for studying methionine oxidation. Myosin is the most abundant protein distributed in skeletal muscles and plays an important role in muscle contraction and metabolism due to the existence of a motor domain and an actin-binding site ( Rayment ., 1993 ; Tokunaga ., 1987 ). Thus, myosin protein has been widely used in studies on muscle protein proteolysis, denaturation, and oxidation, as well as on its relationship with meat quality and carcass trait in livestock ( Coirault ., 2007 ; Hanan and Shaklai, 1995 ; Kim , 2013 ; Lund ., 2008 ). In our previous report, we established that LC-MS/MS was a useful approach to the analysis of myosin isoforms from bovine skeletal muscle. LC-MS/MS can also be used to assess methionine oxidation by identification of mass modification ( Bernevic ., 2011 ; Kim ., 2015 ). Therefore, the purpose of this study was to investigate the oxidized methionine of porcine myosin using proteomic technology.
Materials and Methods
- Sample preparation
Three castrated pigs ((Landrace × Yorkshire) × Duroc) were slaughtered at the body weight of 108±2.5kg (182 d of age) by electrical stunning in a commercial slaughter house. Immediately after slaughter, three muscles ( longissimus thoracis , LT; psoas major , PM; semimembranosus , SM) were taken from each pig within 45 min postmortem. Muscle samples were soaked in lysis buffer consisting of 7 M urea, 2 M thiourea, 1% (w/v) DTT, 4% (w/v) CHAPS, and 0.03 M Tris, and subsequently transferred to a laboratory. Samples were homogenized using a polytron homogenizer (IKA Labortechnik T25-B, Malaysia) at 3,000 rpm for 25 s. The homogenates were centrifuged at 15,000 g at 4℃ for 60 min. Eluted fractions were precipitated by trichloroacetic acid/acetone precipitation and the pellets were dissolved in a lysis buffer. The protein concentration was adjusted to 1.0 mg/mL by using the Bradford (1976) method with bovine serum albumin as a standard. In-solution digestion was conducted using trypsin (Promega, UK). The protein sample was digested in a trypsin solution (2.5 ng/μL) in 50 mM NH 4 HCO 3 buffer (pH 7.8) at 37℃ for 12 h.
- LC-MS/MS and identification of protein and peptides
Protein digests were analyzed using a nano-LC and LTQ mass spectrometer (Applied Biosystems, USA). Samples were injected into a 75 μm × 150 mm column (Proxeon, Denmark) and the capillary column was slurry packed inhouse with 5 μm and 100 Å pore-size Magic C18 stationary phase (Michrom Bioresources, USA). For the LC separation, mobile phase A was 0.1% formic acid in deionized water and mobile phase B was 0.1% formic acid in acetonitrile. Peptides were separated using 75 min gradients (5-35% B for 50 min, 40-60% B for 20 min and 60-80% B for 5 min) and the flow rate was maintained at 300 μL/min after splitting. Mass spectra were acquired from m/z 400-1800 followed by MS/MS scans, and each MS/MS scan acquired was an average of one microscan on the LTQ. The temperature of the ion transfer tube was controlled at 200℃ and the spray was 1.5-2.0 kV. The normalized collision energy was set at 35% for the MS/MS. Peptide sequences were identified via the NCBInr ( Sus scrofa ) database, searching for a peak list using the MASCOT algorithm (Matrix Science, UK). The database search criteria were as follows: fixed modification, no; variable modification, oxidized at methionine residues; maximum allowed missed cleavage, 1; peptide MS tolerance, 100 ppm; fragment MS tolerance, 0.1; peptides resulting from trypsin digest were considered. The ion score was -10×log(P), where P is the probability that the observed match is a random event. Individual ion scores of >39 indicated identity or extensive homology ( p <0.05). All of the identified peptides with ion scores 39 were deleted from the list.
Results and Discussion
- Distribution of myosin isoforms in porcine skeletal muscle
Four myosin isoforms (myosin-1, -2, -4, and -7) were identified in porcine LT, PM, and SM muscles by LC-MS/MS analysis ( Table 1 ). The number of queries matched to the amino acid sequences of myosin isoforms ranged from 72 to 237 and the mean value was 157.9. The average of sequence coverage was 38.0%. It is necessary to check the unique peptides identified from each myosin isoform because of high homology among these isoforms. In the present study, 7.3-13.3 of the unique peptide corresponding to each isoform was detected from three porcine muscles. Although it is impossible to quantitatively analyze the myosin isoforms from the results of this study, we can confirm which isoforms were distributed in the muscle samples. As presented in Table 2 , each unique peptide can be conclusive proof of identification of each myosin isoform. Moreover, these peptides were detected in all three porcine muscles (LT, PM, and SM). Commonly expressed peptides in two or more myosin isoforms supported the distribution of specific myosin isoforms ( Table 3 ). All queries except for DLEEATLQHEATAAALR were peptides composed of myosin-1. These queries were also observed as part of myosin-2 or -4, whereas only four queries were peptides composed of myosin-7.
Myosin isoforms identified from three porcine muscles by LC-MS/MS
PPT Slide
Lager Image
a)Accession numbers were taken from the NCBI database. b)Theoretical molecular weight. c)Peptide size (amino acids) recorded in NCBI database. d)LT, longissimus thoracis; PM, psoas major; SM, semimembranosus.
Unique peptides of each myosin isoform identified from three porcine muscles
PPT Slide
Lager Image
a)Individual ion score > 39 indicates identity (p<0.05).
Queries expressed in common in two or more myosin isoforms identified from three porcine muscles
PPT Slide
Lager Image
a)Individual ion score > 39 indicates identity (p<0.05).
In our previous report, three myosin isoforms (myosin-1, -2, and -7) were identified from bovine longissimus muscle by analysis of LC-MS/MS ( Kim, 2014 ). However, additional analyses were needed because it was not confirmed whether myosin-4 existed in this muscle. Although no unique queries of myosin-4 were detected, some queries were identified as peptides expressed in common in two or more isoforms including myosin-4. A number of previous reports that studied myosin isoforms using various technologies, such as electrophoresis, histochemistry, immunohistochemistry, immunoblotting, and RT-PCR in various breeds of pigs, supported the clain that there are four myosin isoforms in porcine skeletal muscle ( Abreu ., 2006 ; Chang ., 2003 ; Kim ., 2014 ; Lefaucheur ., 2002 ). It has been observed that the composition of myosin-7 (myosin heavy chain I/slow) in porcine skeletal muscle was the lowest among the myosin isoforms at the mRNA and protein levels ( Choi ., 2007 ; Park ., 2009 ). In this study, myosin-7 had lower values in matched queries and in sequence coverage compared to the others.
- Identification of oxidized methionine
Five methionine-oxidized peptides were detected among 836 trypsin-digested peptides, as shown in Table 4 . SM muscle had all of the methionine-oxidized peptides, whereas PM and LT had one and two, respectively, and these oxidized peptides had one or two oxidized methionines. Trypsin-digested peptides generally had a methionine, and their oxidation could be identified by mass modification. Peptide IEDEQALAMQLQK, which corresponded to residues 1098-1110 of myosin-1 (or residues 1096-1108 of myosin-4), had an oxidized methionine. The MS/MS spectra of this peptide showed 16 amu of mass modification in residue 106 ( Fig. 1 ). The mass of y(5) corresponding to 1106 MQLQK 1110 was m/z 647.8071 ( Fig. 1A ), whereas the spectrum of oxidized peptide corresponding to 1106 MQLQK 1110 was observed at 16.0 amushift (m/z 663.8065) ( Fig. 1B ). The mass of b(9) also showed the same mass shift (m/z 1002.1191 to m/z 1018.1185) in 1098 IEDEQALAM 1106 ( Fig. 1 ).
Methionine-oxidized peptides of myosin isoform identified from three major porcine muscles by LC-MS/MS
PPT Slide
Lager Image
a)LT, longissimus thoracis; PM, psoas major; SM, semimembranosus. b)Individual ion score > 39 indicates identity (p<0.05). c)Myosin isoform (residues). M on the bar indicates oxidized methionine.
PPT Slide
Lager Image
LC-MS/MS spectra of precursor ions m/z 759.86 (A) and m/z 767.79 (B) corresponding to residues 1098-1110 (IEDEQALAMQLQK) of myosin-1.
MFLWMVTR, which corresponded to residues 438-445, had two methionines (residues 438 and 442). As presented in Fig. 2 , the methionine corresponding to residue 438 was identified as methionine sulfoxide (the addition of one oxygen), whereas another methionine (residue 442) did not oxidize. The masses of b(2) and b(3) from non-modified peptides ( Fig. 2A ) were m/z 279.3774 and m/z 392.5350, respectively, whereas those from modified peptides ( Fig. 2B ) were m/z 295.3768 and m/z 408.5344, respectively. Thus, 16 amu of mass shift was confirmed by the mass of b(2) and b(3). The b(7) corresponding to 438 MFLWMVT 445 indicates that methionine on residue 442 did not oxidize due to the mass shift, was 16.0amu (m/z 910.1750 to m/z 926.1754). Moreover, there was no mass shift of y(4) between non-oxidized peptide (m/z 506.6395) and oxidized peptide (m/z 506.6395).
PPT Slide
Lager Image
LC-MS/MS spectra of precursor ions m/z 542.94 (A) and m/z 551.16 (B) corresponding to residues 438-445 (MFLWMVTR) of myosin-1.
Two methionine oxidations were found in SAETEKEMANMKEEFEK that corresponded to residues 846-862 of myosin-1 (or residues 844-860 of myosin-4) ( Fig. 3 ). The mass difference of b(11) between non-oxidized peptide (m/z 1223.3535) and oxidized peptide (m/z 1255.3523) was 32.0amu. The mass shift of b(11) indicated that two oxygens were added to this peptide, and three cases are predictable as follows: the methionine on residue 853 oxidized to the sulfone form, another methionine (on residue 856) formed a methionine sulfone, and both of the methionines oxidized as sulfoxide forms. The y(8) corresponding to 855 NMKEEFEK 862 showed 16.0 amu of mass shift, while 32.0 amu of mass shift was found for the y 0 (10) corresponding to 853 MANMKEEFEK 862 .
PPT Slide
Lager Image
LC-MS/MS spectra of precursor ions m/z 678.28 (A) and m/z 689.06 (B) corresponding to residues 846-862 (SAETEKEMANMKEEFEK) of myosin-1.
It is generally accepted that protein-bound methionine easily forms methionine sulfoxide by reacting with peroxides, and methionine sulfone may also often be formed ( Vogt, 1995 ). In the present study, only methionine sulfoxide was detected. Very little oxidized methionine was identified in porcine skeletal muscle, and this may be due to the early collection of muscle samples (within 45 min postmortem). In the muscle-food system, myofibril proteins are affected by various oxidants during processing and storage ( Martinaud ., 1997 ). In addition, the oxidation of methionine and other amino acids can be affected by neighboring amino acids ( Ghesquiere ., 2011 ). Methionines surrounded by acidic amino acids, such as aspartic acid (D) and glutamic acid (E), or those surrounded by alanine (A), threonine (T), and serine (S), are susceptible for oxidation. In the present study, there were A or T nearby oxidized methionines on residues 853, 1071 and 106 of myosin-1.
It is known that the general oxidative changes in proteins are protein cross-linking, modifications of amino acid side chains and protein fragmentation caused by the formation of disulfide bonds, hydroperoxide, carbonyl, sulfoxide, and sulfone groups, as reported by Lund . (2011) . These oxidants lead not only to reducing the water-holding capacity and the tenderness of meat, but also the deterioration of protein functionality, such as in hydrophobicity, conformation, and solubility ( Lund ., 2007 ; Rowe ., 2004 ). The effect of methionine oxidation on cellular signaling was the main focus in previous reports ( Erickson ., 2008 ; Hardine ., 2009 ). However, little attention was paid to the impact of methionine oxidation in food systems.
All of the oxidized peptides identified in porcine skeletal muscles were commonly-expressed peptides in two or three myosin isoforms, while there was no unique peptide expressed in one myosin isoform among the oxidized peptides. In particular, no peptides of myosin-7 were detected as oxidized peptides, regardless of whether they were unique or common peptides. This may be due to the different compositions of the myosin isoforms of the muscle sample. The content of myosin-7 is the lowest among the myosin isoforms in porcine skeletal muscles ( Choi ., 2007 ; Park ., 2009 ). In this study, we did not quantified each myosin isoform, but it was estimated that the content of myosin-7 was lower than the others because of the lowest number of matched queries, as shown in Table 1 . Protein oxidation can occur by chemicals and equipment during analytical steps ( Chen and Cook, 2007 ; Liu ., 2013 ). Nevertheless, LC-MS/MS is better suited for studying protein oxidation than two-dimensional gel electrophoresis ( Ghesquière and Gevaert, 2014 ). Liu . (2013) presented the accurate determining method for methionine oxidation by stable isotope labeling and LC-MS analysis. In the present study, although overestimation of oxidized methionine level was not excluded, oxidized methionine can be detected from porcine skeletal muscles by LC-MS/MS analysis.
Five peptides with one or two oxidized methionines were detected among 836 queries of myosin isoforms from porcine skeletal muscles, and these were identified by LC-MS/MS. All oxidized methionines were identified in the methionine sulfoxide form by an oxygen modification (16 amu-shift), while methionine sulfone was not observed in the present study. The LC-MS/MS approach can also allow the analysis of methionine oxidation on specific residues and specific myosin isoforms in porcine skeletal muscle.
National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2015R1A1A3A04001313).
Abreu E. , Quirox-Rothe E. , Mayoral A. I. , Vivo J. M. , Robina Á. , Guillén M. T. , Agüera E. , Rivero J. L. (2006) Myosin heavy chain fibre types and fibre sizes in nulliparous and primiparous ovariectomized Iberian sows: Interaction with two alternative rearing systems during the fattening period Meat Sci. 74 359 - 372    DOI : 10.1016/j.meatsci.2006.04.002
Armenteros M. , Heinonen M. , Ollilainen V. , Toldrá F. , Estévez M. (2009) Analysis of protein carbonyls in meat products by using the DNPH method, fluorescence spectroscopy and liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) Meat Sci. 83 104 - 112    DOI : 10.1016/j.meatsci.2009.04.007
Bernevic B. , Petre B. A. , Galetskiy D. , Werner C. , Wicke M. , Schellander K. , Przybylski M. (2011) Degradation and oxidation postmortem of myofibrillar proteins in porcine skeleton muscle revealed by high resolution mass spectrometric proteome analysis Int. J. Mass Spectrom. 305 217 - 227    DOI : 10.1016/j.ijms.2010.11.010
Bradford M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding Anal. Biochem. 72 248 - 254    DOI : 10.1016/0003-2697(76)90527-3
Chang K. C. , Costa N. , Blackley R. , Southwood O. , Evans G. , Plastow G. , Wood J. D. , Richardson R. I. (2003) Relationships of myosin heavy chain fibre types to meat quality traits in traditional and modern pigs Meat Sci. 64 93 - 103    DOI : 10.1016/S0309-1740(02)00208-5
Chen M. , Cook K. D. (2007) Oxidation artifacts in the electrospray mass spectrometry of Aβ peptide Anal. Chem. 79 2031 - 2036    DOI : 10.1021/ac061743r
Choi Y. M. , Ryu Y. C. , Kim B. C. (2007) Influence of myosin heavy- and light chain isoforms on early postmortem glycolytic rate and pork quality Meat Sci. 76 281 - 288    DOI : 10.1016/j.meatsci.2006.11.009
Coirault C. , Guellich A. , Barbry T. , Samuel J. L. , Riou B. , Lecarpentier Y. (2007) Oxidative stress of myosin contributes to skeletal muscle dysfunction in rats with chronic heart failure Am. J. Physiol. Heart Circ. Physiol. 292 H1009 - H1017
Daneshvar B. , Frandsen H. , Artrup H. , Dragsted L. O. (1997) γ-Glutamyl semialdehyde and α-amino-adipic semialdehyde: Biomarkers of oxidative damage to proteins Biomarkers 2 117 - 123    DOI : 10.1080/135475097231841
Decker E. A. , Xiong Y. L. , Calvert J. T. , Crum A. D. , Blanchard S. P. (1993) Chemical, physical, and functional-properties of oxidized turkey white muscle myofibrillar proteins J. Agric. Food Chem. 41 186 - 189    DOI : 10.1021/jf00026a007
Erickson J. R. , Joiner M. L. , Guan X. , Kutschke W. , Yang J. , Oddis C. V. , Bartlett R. K. , Lowe J. S. , O’Donnell S. E. , Aykin-Burns N. , Zimmerman M. C. , Zimmerman K. M , Ham A. J. , Weiss R. M. , Spitz D. R. , Shea M. A. , Colbrau R. J. , Mohler P. J. , Anderson M. E. (2008) A dynamic pathway for calcium-independent activation of CaMKII by methionine oxidation Cell 133 462 - 474    DOI : 10.1016/j.cell.2008.02.048
Estévez M. , Ollilainen V. , Heinonen M. (2009) Analysis of protein oxidation markers α-aminoadipic and γ-glutamic semialdehydes in food proteins using liquid chromatography (LC)-electrospray ionization (ESI)-multistage tandem mass spectrometry (MS) J. Agric. Food Chem. 57 3901 - 3910    DOI : 10.1021/jf804017p
Garrison W. M. (1987) Reaction mechanisms in the radiolysis of peptides, polypeptides, and proteins Chem. Rev. 87 381 - 398    DOI : 10.1021/cr00078a006
Ghesquière B. , Gevaert K. (2014) Proteomics methods to study methionine oxidation Mass Spectrom. Rev. 33 147 - 156    DOI : 10.1002/mas.21386
Ghesquière B. , Jonckheere V. , Colaert N. , Van Durme J. , Timmerman E. , Goethals M. , Schymkowita J. , Rousseau F. , Vandekerckhove J. , Gevaert K. (2011) Redox proteomics of protein-bound methionine oxidation Mol. Cell Proteomics M110.006866 10
Hanan T. , Shaklai N. (1995) Peroxidative interaction of myoglobin and myosin Eur. J. Biochem. 233 930 - 936    DOI : 10.1111/j.1432-1033.1995.930_3.x
Hardine S. C. , Larue C. T. , Oh M. H. , Jain V. , Huber S. C. (2009) Coupling oxidative signals to protein phosphorylation via methionine oxidation in Arabidopsis Biochem. J. 422 305 - 312    DOI : 10.1042/BJ20090764
Jongberg S. , Gislason N. E. , Lund M. N. , Skibsted L. H. , Waterhouse A. L. (2011) Thiol-quinone adduct formation in myofibrillar proteins detected by LC-MS J. Agric. Food Chem. 59 6900 - 6905    DOI : 10.1021/jf200965s
Kim G. D. (2014) Analysis of myosin heavy chain isoforms from logissimus toracis muscle of Hanwoo steer by electrophoresis and LC-MS/MS Korean J. Food Sci. An. 34 656 - 664    DOI : 10.5851/kosfa.2014.34.5.656
Kim G. D. , Jeong J. Y. , Yang H. S. , Joo S. T. (2015) Analysis of oxidized methionione in myosin isoforms of porcine longissimus thoracis muscle at 24 h postmortem Proceed. 61st Int. Cong. Meat Sci. Technol. Clermont-Ferrand, France 163 -
Kim G. D. , Ryu Y. C. , Jeong J. Y. , Yang H. S. , Joo S. T. (2013) Relationship between pork quality and characteristics of muscle fibers classified by the distribution of myosin heavy chain isoforms J. Anim. Sci. 91 5525 - 5534    DOI : 10.2527/jas.2013-6614
Kim G. D. , Ryu Y. C. , Jo C. , Lee J. G. , Yang H. S. , Jeong J. Y. , Joo S. T. (2014) The characteristics of myosin heavy chain-based fiber types in porcine longissimus dorsi muscle Meat Sci. 96 712 - 718    DOI : 10.1016/j.meatsci.2013.09.028
Lefaucheur L. , Ecolan P. , Plantard L. , Gueguen N. (2002) New insights into muscle fiber types in the pig J. Histochem. Cytochem. 50 719 - 730    DOI : 10.1177/002215540205000513
Levine R. L. (1984) Mixed-function oxidation of histidine residues Methods Enzymol. 107 370 - 376
Liu H. , Ponniah G. , Neill A. , Patel R. , Andrien B. (2013) Accurate determination of protein methionine oxidation by stable isotope labelling and LC-MS analysis Anal. Chem. 85 11705 - 11709    DOI : 10.1021/ac403072w
Lund M. N. , Heinonen M. , Baron C. P. , Estévez M. (2011) Protein oxidation in muscle foods: A review Mol. Nutr. Food Res. 55 83 - 95    DOI : 10.1002/mnfr.201000453
Lund M. N. , Lametsch R. , Hviid M. S. , Jensen O. N. , Skibsted L. H. (2007) High-oxygen packaging atmosphere influences protein oxidation and tenderness of porcine longissimus dorsi during chill storage Meat Sci. 77 295 - 303    DOI : 10.1016/j.meatsci.2007.03.016
Lund M. N. , Luxford C. , Skibsted L. H. , Davies M. J. (2008) Oxidation of myosin by heam proteins generates myosin radicals and protein cross-links Biochem. J. 410 565 - 574    DOI : 10.1042/BJ20071107
Martinaud A. , Mercier Y. , Marinova P. , Tassy C. , Gatellier P. , Renerre M. (1997) Comparison of oxidative processes on myofibrillar proteins from beef during maturation and by different model oxidation systems J. Agric. Food Chem. 45 2481 - 2487    DOI : 10.1021/jf960977g
Oh-Ishie M. , Ueno T. , Maeda T. (2003) Proteomic method detects oxidatively induced protein carbonyls in muscles of a diabetes model Otsuka Long-Evans Tokushima fatty (Oletf) rat Free Radical Biol. Med. 34 11 - 22    DOI : 10.1016/S0891-5849(02)01239-X
Oliver C. N. , Ahn B. W. , Moerman E. J. , Goldstein S. , Stadtman E. R. (1987) Age-related changes in oxidized proteins J. Biol. Chem. 262 5488 - 5491
Park S. K. , Gunawan A. M. , Scheffler T. L. , Grant A. L. , Gerrard D. E. (2009) Myosin heavy chain isoform content and energy metabolism can be uncoupled in pig skeletal muscle J. Anim. Sci. 87 522 - 531
Rayment I. , Rypniewski W. R. , Schmidt-Base K. , Smith R. , Tomchick D. R. , Benning M. M. , Winkelmann D. A. , Wesenberg G. , Holden H. M. (1993) Three dimensional structure of myosin subfragment-1: A molecular motor Science 261 50 - 58    DOI : 10.1126/science.8316857
Rowe L. J. , Maddock K. R. , Lonergan S. M. , Huff-Lonergan E. (2004) Influence of early postmortem protein oxidation on beef quality J. Anim. Sci. 82 785 - 793
Shacter E. (2000) Quantification and significance of protein oxidation in biological samples Drug Met. Rev. 32 307 - 326    DOI : 10.1081/DMR-100102336
Soladoye O. P. , Juárez M. L. , Aalhus J. L. , Shand P. , Estévez M. (2015) Protein oxidation in processed meat: Mechanisms and potential implications on human health Compr. Rev. Food Sci. F. 14 106 - 122    DOI : 10.1111/1541-4337.12127
Stadtman E. R. (1993) Oxidation of free amino acids and amino acid residues in proteins by radiolysis and by metal-catalyzed reactions Annu. Rev. Biochem. 62 797 - 821    DOI : 10.1146/
Stadtman E. R. , Berlett B. S. (1988) Fenton chemistry revisited: Amino acid oxidation Basic Life Sci. 49 131 - 136
Stadtman E. R. , Levine R. L. (2003) Free radical-mediated oxidation of free amino acids and amino acid residues in proteins Amino Acids 25 207 - 218    DOI : 10.1007/s00726-003-0011-2
Tokunaga M. , Sutoh K. , Toyoshima C. , Wayabashi T. (1987) Location of the ATPase site of myosin determined by three-dimensional electron microscopy Nature 329 635 - 638    DOI : 10.1038/329635a0
Vogt W. (1995) Oxidation of methionyl residues in proteins: Tools, targets, and reversal Free Radic. Biol. Med. 18 93 - 105    DOI : 10.1016/0891-5849(94)00158-G
Winterbourn C. C. (1990) Oxidative reactions of hemoglobin Methods Enzymol. 186 265 - 272
Xiong Y. L. , Decker E. , Faustman C. , Clemente J. L. B. (2000) Antioxidant in muscle foods Wiley Chichester, UK Protein oxidation and implications for muscle food quality 85 - 111