Application of Gel-based Proteome Analysis Techniques to Studying Post-mortem Proteolysis in Meat

  • 투고 : 2003.12.02
  • 심사 : 2004.05.11
  • 발행 : 2004.09.01


This study was conducted to evaluate the possible application of 2 D-SDS-PAGE (2 DE)-based proteome analysis techniques to the assessment of extreme proteolysis in postmortem skeletal muscle. Eight Hanwoo longissimus muscles were incubated immediately after slaughter for 24 h at 5$^{\circ}C$, 15$^{\circ}C$ or 36$^{\circ}C$. Warner Bratzler (WB)-shear force and ultrastructural configuration were determined at 24 h, and rate of proteolysis to 24 h was determined by 1 D-SDS-PAGE (1 DE) and 2 DE. In addition, tentative protein identification was performed from peptide mass fingerprints of MALDI-ToF analysis of major protein groups on 2 DE profiles. The result showed that although ultrastructural configuration was similar between the 5$^{\circ}C$ and 36$^{\circ}C$ treatments, meat at 5$^{\circ}C$ had higher WBshear force (approximately 5 kg greater). A higher rate of protein degradation at 36$^{\circ}C$ was observed based on Troponin-T degradation, 1 DE, and 2 DE analysis. This indicates that proteolysis during the early postmortem period was a significant determinant of shear force at 24 h. Little difference in proteolysis between 5$^{\circ}C$ and 15$^{\circ}C$ treatments was found based on classic 1 DE profile assessment. Meanwhile, considerable differences in the 2 DE profiles between the two treatments were revealed, with substantially higher rate of proteolysis at 15$^{\circ}C$ compared to 5$^{\circ}C$. Nuclease treatment improved 2 DE profile resolution. 400 ${\mu}$g and 600 ${\mu}$g of sample loading appeared to be appropriate for 24 cm pH 3-10 and pH 5-7 IPG strips, respectively. Protein detection and quantification of the 5$^{\circ}C$, 15$^{\circ}C$ and 36$^{\circ}C$ 2 DE profiles revealed 78, 163 and 232 protein spots respectively that were differentially modified in terms of their electrophoretic properties between approximately pI 5.3-7.7 with the molecular weight range of approximately 71-12 kDa. The current results demonstrated that 2 DE was a superior tool to 1 DE for characterising proteolysis in postmortem skeletal muscle.


  1. Bailey, A. J. and N. D. Light. 1989. Connective Tissue in Meat and Meat Products. Elsevier Science Publisher, London.
  2. Hopkins, D. L. and J. M. Thompson. 2001. Inhibition of protease activity Part 1. The effect on tenderness and indicators of proteolysis in ovine muscle. Meat Sci. 59:175-185.
  3. Hwang, I. H. and J. M. Thompson. 2001b. The effect of time and type of electrical stimulation on the calpain system and meat tenderness in beef longissimus dorsi muscle. Meat Sci. 58:134-144.
  4. Hwang, I. H. and J. M. Thompson. 2002. A Technique to Quantify the Extent of Postmortem Degradation of Meat Ultrastructure. Asian-Aust. J. Anim. Sci. 15:111-116.
  5. Hwang, I. H., C. E. Devine and D. L. Hopkins. 2003a. The biochemical and physical effects of electrical stimulation on beef and sheep meat tenderness. Meat Sci. 65:677-691.
  6. Hwang, I. H. 2004. Proteomics Approach in Meat Science: A model study for meat color and drip loss. Food Sci. Biotech. 13:208-214.
  7. Hwang, I. H., B. Y. Park, S. H. Cho and J. M. Lee. 2004. Effects of muscle shortening and proteolysis on Warner-Bratzler shear force in beef longissimus and semitendinosus. Meat Sci. 68:497-505.
  8. Koohmaraie, M. 1992. Effect of pH, temperature and inhibitors on autolysis and catalytic activity of bovine skeletal muscle ${\mu}$-calpain. J. Anim. Sci. 70:3071-3080.
  9. Liebler, D. C. and J. R. Yates. 2002. Introduction to Proteomics-Tools for the New Biology. Humana Press Inc., New Jersey.
  10. Moon, S. S., I. H. Hwang, S. K. Jin, J. G. Lee, S. T. Joo and G. B. Park. 2003. Carcass traits determining quality and yield grades of Hanwoo steers. Asian-Aust. J. Anim. Sci. 16:1049-1054.
  11. Parker, K. C., J. I. Garrels, W. Hines, E. M. Butler, A. H. McKee, D. Patterson and S. Martin. 1998. Identification yeast proteins from two-dimensional gels: working out spot crosscontamination. Electrophoresis. 19:1920-1932.
  12. Rabilloud, T. 1996. Solubilization of proteins for electrophoretic analyses. Electrophoresis. 17:813-829.
  13. Scopes, R. K. 1994. Protein purification: principles and practice ($3^{rd}$ Ed). Springer-Verlag, New York.
  14. Westerblad, H. and D. G. Allen. 1993. The influence of intracellular pH on contraction, relaxation and [$Ca^{2+}$]I in intact single fibres from mouse muscle. J. Physi. 466:611-628.
  15. Westermeier, R. and T. Naven. 2002. Proteomics in practice-a laboratory manual of proteome analysis. Wiley-VCH Verlag-GmbH, Weinheim.
  16. Yan, J. X., R. A. Harry, R. Wait, S. Y. Welson, P. W. Emery, V. R. Preedy and M. J. Dunn. 2001. Separation and identification of rate skeletal muscle proteins using two-dimensional gel electrophoresis and mass spectrometry. Proteomics. 1:424-434.
  17. Olson, D. G., F. C. Jr. Parrish, W. R. Dayton and D. E. Goll. 1977. Effect of postmortem storage and calcium activated factor on myofibrillar proteins of bovine skeletal muscle. J. Food. Sci. 42:117-124.
  18. Jeacocke, R. E. 1993. The concentrations of free magnesium and free calcium ions both increase in skeletal muscle fibres entering rigor mortis. Meat Sci. 35:27-45.
  19. Koohmaraie, M. 1996. Biochemical factors regulating the toughening and tenderization processes of meat. Meat Sci. 43:S193-S201.
  20. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 227:680-685.
  21. Pennington, S. R. and M. J. Dunn. 2001. Proteomics from protein sequence to function. Springer-Verlag, New York.
  22. Ho, C. Y., M. H. Stromer, G. Rouse and R. M. Robson. 1997. Effects of electrical stimulation and postmortem storage on changes in titin, nebulin, desmin, troponin-T, and muscle ultrastructure in Bos indicus crossbred cattle. J. Anim. Sci. 75:366-376.
  23. Morzel, M., V. Bagnic-Verrez, E. K. Arendt and J. Fleurence. 2000. Use of Two-Dimensional Electrophoresis to evaluate proteolysis in Salmon (Salmo salar) muscle as affected by a lactic fermentation. J. Agric. Food Chem. 48:239-244.
  24. Devine, C. E., S. R. Payne, B. M. Peachey, T. E. Lowe, J. R. Ingram and C. J. Cook. 2002. High and low rigor temperature effects on sheep meat tenderness and ageing. Meat Sci. 60:141-146
  25. Lilley, K. S., A. Razzaq and P. Dupree. 2001. Two-dimensional gel electrophoresis: recent advances in sample preparation, detection and quantitation. Current Opin. Chem. Biolog. 6:46-50.
  26. Tornberg, E. 1996. Biophysical aspects of meat tenderness. Meat Sci. 43:S175-S191.
  27. Isfort, R. J. 2002. Review-Proteomic analysis of striated muscle. J. Chromatogr. 771:155-165.
  28. Campbell, A. M. and L. J. Heyer. 2003. Genomics, Proteomics and Bioinformatics. Courier Book Companies, New York,
  29. Claeys, E., L. Uytterhaegen, D. Demeyer and S. DeSmet. 1994. Beef myofibrillar protein salt solubility in relation to tenderness and proteolysis. In: Proc. 40th Int. Congr. Meat Sci. and Technol., The Hague, The Netherlands, p. SIVB 09.
  30. Hwang, I. H. and J. M. Thompson. 2003b. Effects of pH early postmortem on meat quality in beef longissimus. Asian-Aust. J. Anim. Sci. 16:1218-1223.
  31. Lametsch, R. and E. Bendixen. 2001. Proteome analysis applied to meat science: Characterizing postmortem changes in porcine muscle. J. Agric. Fd. Chem. 49:4531-4537.
  32. Locker, R. H. 1960. Degree of muscular contraction and as a factor in the tenderness of beef. Food Res. 25:304-307.
  33. Devine, C. E., N. M. Wahlgren and E. Tornberg. 1999. Effect of rigor temperature on muscle shortening and tenderisation of restrained and unrestrained beef m. longissimus thoracis et lumborum. Meat Sci. 51: 61-72.
  34. Davey, C. L. and K. V. Gilbert. 1966. Studies in meat tenderness II. Proteolysis and the aging of beef. J. Fd. Sci. 31:135-140.
  35. Hopkins, D. L., P. J. Littlefield and J. M. Thompson. 2000. A research note on factors affecting the determination of myofibrillar fragmentation, Meat Sci. 56:19-22.
  36. Dransfield, E. 1994. Optimisation of tenderisation, ageing and tenderness. Meat Sci. 36: 105-121.
  37. Geesink, G. H., M. H. D. Mareko, J. D. Morton and R. Bickerstaffe. 2001. Effects of stress and high voltage electrical stimulation on tenderness of lamb m. Longissimus. Meat Sci. 57:265-271.
  38. Wheeler, T. L., S. D. Shackelford and M. Koohmaraie. 2000. Relationship of beef longissimus tenderness classes to tenderness of gluteus medius, semimembranosus, and biceps femoris. J. Anim. Sci. 78:2856-2861.
  39. Min, J. S., I. S. Kim, Y. T. Yoon and M. Lee. 2002. Real effect of pH on CIE L*, a* and b* of loins during 24 h chilling of beef carcasses. Asian-Aust. J. Anim. Sci. 15:279-282.
  40. Wheeler, T. D. and M. Koomaraie. 1999. The extent of proteolysis is independent of sarcomere length in lamb longissimus and psoas major. J. Anim. Sci. 77:2444-2451.
  41. Hwang, I. H. and J. M. Thompson. 2001a. The interaction between pH and temperature decline early postmortem on the calpain system and objective tenderness in electrically stimulated beef longissimus dorsi muscle. Meat Sci. 58:167-174.
  42. Bendall, J. R. 1973. Postmortem changes in muscle. In: (Ed. G. H. Bourne). The Structure and Function of Muscle. pp. 244-306. Academic Press, New York.
  43. Pollard, J. W. 1994. Two-Demensional Polyacrylamide Gel Electrophoresis of Proteins. In: (Ed. J. M. Walker). Methods in molecular biology-basic protein and peptide protocols. pp. 73-85. Humana Press, New Jersey.

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