Article pp.75-78 du Vol.23 n°1 (2003)

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SCIENCES DES ALIMENTS, 23(2003) 75-78

FOCUS : JSMTV

Proteome analysis applied to the study of muscle development and sensorial qualities

of bovine meat

J. Bouley, C. Chambon*, B. Picard ¹

INTRODUCTION

The origin and the evolution of bovine muscle characteristics implied in meat sensory qualities have been the subject of many studies. These have shown that bovine muscle is differentiated from the last trimester of foetal life but presents a large plasticity during postnatal life. Thus, the muscle characteristics, in particular those of the fibres, constantly evolve during the life of the ani- mal and can be modified under the influence of various breeding factors such as nutrition. The genetic type of animals also conditions the muscle properties considerably. In particular, it has been clearly shown that a high muscle development, from monogenic (double-muscled genotype) or polygenic (divergent lineages) origin is accompanied by particular characteristics. Muscles of these animals contain a higher total number of fibres, a greater proportion of fast glycolytic fibres following on a greater proliferation of second cell generation (DEVEAUX et al., 2001). Thus, these muscles are more glycolytic and have a lower collagen and intra-muscular lipid contents than muscles of non hypertro- phied animals. These characteristics act in favour of improvement of the meat tenderness but against improvement of its flavour. It appears important to identify the origin of these particular characteristics associated with high muscle development to understand the mechanisms of regulation implied and to be able to modulate them by breeding factors in order to master both the tenderness and the flavour of beef meat. To this end we chose to develop proteome analysis that permits the simultaneous study of hundreds of proteins to identify muscle hypertrophy markers. The aim of this work was to demonstrate the fea- sibility of separation of bovine muscle proteins by two-dimensional gel electrophoresis (2DE) and to apply it to the study of muscle hypertrophy.

1. Unité de Recherches sur les Herbivores. *Station de Recherches sur la viande.

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76 Sci. Aliments 23(1), 2003 J. Bouley et al.

MATERIAL AND METHODS

The Semitendinosus muscle (ST) (mixed fast glycolytic) was sampled on different types of bovines in the hour following slaughter, it was frozen in liquid nitrogen and stored at –80˚C. The following animals were used for this study:

Model 1, 100-day-old Belgian Blue foetuses double-muscled homozygotes (BDM) and non double-muscled homozygotes (BNDM), Holsteins (H) (non double-muscled); Model 2, Belgian Blue bulls of 22 months homozygotes (BDM), double-muscled heterozygotes (HDM) and non double-muscled homozygotes (BNDM); Model 3, bulls of 19 months from two divergent lineages of high (++) or low (--) muscle development. The frozen muscle samples (40 mg) were ground in 1 ml of lysis buffer and analysed by 2DE (YAN et al., 2001). Iso-electric focus- ing (Multiphor, Amersham Pharmacia Biotech) was achieved on 3-10 or 4-7 pH gradients (IPG, 13 cm) (Amersham Pharmacia Biotech) and the second dimen- sion (Hoefer system), on the SDS-PAGE gels (11% T, 2.6% C). For the analytic (50 µg) and preparative (0.2 mg to 1 mg) gels, proteins were visualised respec- tively by silver nitrate coloration and by Coomassie Blue R 250 staining. Gels were analysed and compared to each other using Image Master 2D Elite soft- ware (Amersham). Proteins of interest were excised from the gels and were pre- pared for analysis by MALDI-tof (Voyager-DE pro, PE Biosystems) according to the protocol “Montage In Gel Digest₉₆ Kit (Millipore)”. Mass spectra were sub- mitted to two mass fingerprinting databases: Profound and Mascot.

RESULTS / DISCUSSION

Two-dimensional gel electrophoresis applied to bovine ST muscle under our conditions allowed about 1000 very resolute spots in the 4-7 pH gradient and roughly 500 spots in the 3-10 pH gradient to be revealed in a molecular mass zone comprised between 15 and 200 kDa (Fig. 1). Contrary to the foetal muscles presenting 80% of proteins in the 4-7 pH gradient zone, the 2DE of adult muscles show a homogeneous distribution of proteins in the 3-10 pH gradient.

However, it is clear that the latter do not offer a satisfactory resolution because of the strong dynamic of muscle protein expression. Indeed, the numerous basic proteins highly expressed limit the visualisation of other less abundant proteins. So most comparative studies are made on 4-7 pH gradients while wai- ting for improvement in basic protein resolution (on IPG, 24 cm). However, the reproducibility of the method shows that on average 90% of spots are revealed on the different gels within the studied models. Recently, two studies permitted generation of skeletal muscle reference maps annotated thanks to the mass spectrometer (YAN et al., 2001, SANCHEZ et al., 2001). Comparison of our results with these maps as well as with those of LAMETSCH and BENDIXEN (2001) reveals very considerable similarities permitting to conclude that this protein separation technique is applicable to the analysis of bovine muscle.

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Proteome analysis applied to the study of muscle development and sensorial qualities of bovine meat 77

Figure 1

Two-dimensional gel electrophoresis of adult ST muscle (1, IPG 3-10 and 2, IPG 4-7) and 100-day foetal ST muscle (3, IPG 3-10 and 4, IPG 4-7).

50 µg of proteins were separated by 2DE and visualised by silver nitrate staining

The gel analysis within the different study models indicates that quantitative and qualitative variations of protein phenotype concern a very low proportion of proteins (below 5%). The most important variability was recovered by compa- ring the ST muscle of adult BDM and BNDM bovines. Out of 26 proteins present in the 4-7 pH gradient, 12 were under-expressed and 14 over-expressed in double-muscled muscles. For example, figure 2 shows one under-expression of the troponin T slow skeletal muscle isoform (TnT slow, N1) in the DM ST muscle and one over-expression of an isoform of the Triosephosphate isomerase (H2).

The under-expression of TnT slow and the over-expression of the Triosephos- phate isomerase isoform are coherent with a more elevated proportion of fast glycolytic fibres (DEVEAUX et al., 2001). On the set of proteins concerned by a variation of expression, several were recovered within the different studied models. Thus, it seems that these proteins variably expressed in the different models could be good markers for muscle hypertrophy.

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78 Sci. Aliments 23(1), 2003 J. Bouley et al.

Figure 2

Portions of 2DE presented in figure 1 (number 2). N1 shows one under-expression of the protein troponin T slow skeletal muscle isoform (TnT slow) in the ST muscle

of adult bovines BDM. H2 shows one over-expression of an isoform of the Triosephosphate isomerase protein in the same muscle.

CONCLUSION

In conclusion, proteomic analysis applied to bovine muscles permits simultaneous study of different classes of proteins (metabolic enzymes, contractile apparatus, chaperones…) and identification of proteins of interest differentially expressed in the studied models. These first results show that it is possible to reveal the potential markers for muscle hypertrophy. In the short term, valida- tion of these markers on different types of muscles and of bovines will be achieved by more classical techniques (RT-PCR, western-blot,…). This approach will also be applied to studies concerning muscle differentiation and also to seek markers for bovine meat tenderness.

REFERENCES H2

N1

Hypertrophy (BDM) Normal (BNDM)

DEVEAUX V., CASSAR MALEK I., PICARD B., 2001. Comparison of contractile characteristics of muscle from Holstein and double-muscled Belgian Blue foetuses.

Comp. Biochem. Physiol. Part A 131 21- 29.

YAN J.X., HARRY R.A., WAIT R., WELSON S.Y., EMERY P.W., PREEDY V.R., DUNN M.J., 2001. Separation and identification of rat muscle proteins using two-dimensional gel electrophoresis and mass spectrometry. Proteomics 1, 424-434.

SANCHEZ J.C., CHIAPPE D., CONVERSET V., HOOGLAND C., BINZ P.A., PAESANO S., APPEL R.D., WANG S., SENNITT M., NOLAN A., CAWTHORNE M.A., HOCHS- TRASSER D.F., 2001. The mouse SWISS- 2D PAGE database: a tool for proteomics study of diabetes and obesity. Proteomics 1, 136-163.

LAMETSH R., BENDIXEN E., 2001. Proteome analysis applied to meat science: charac- terizating post mortem changes in porcine muscle. J. Agric. Food Chem. 49 4531- 4537.