Elsevier

Food Chemistry

Volume 169, 15 February 2015, Pages 65-72
Food Chemistry

Analytical Methods
Bioanalytical methods for the metalloproteomics study of bovine longissimus thoracis muscle tissue with different grades of meat tenderness in the Nellore breed (Bos indicus)

https://doi.org/10.1016/j.foodchem.2014.07.131Get rights and content

Highlights

  • 2D-PAGE efficiently proteins with different grades of meat tenderness.

  • Total protein extraction procedures preserved the metal–protein structure.

  • Pyruvate kinase and albumin were inferred to be related to the phenotypical differences.

  • Those two proteins likely partly explain the variation in the tenderness of their meat.

Abstract

The work describes a metalloproteomics study of bovine muscle tissue with different grades of meat tenderness from animals of the Nellore breed (Bos indicus) based on protein separation by two-dimensional gel electrophoresis, the identification of calcium ions in protein spots by X-ray fluorescence (SR-XRF) and the characterisation of proteins by electrospray ionisation mass spectrometry. Forty (40) specimens were selected and divided into two experimental groups: animals with tough meat (TO) and animals with tender meat (TE). A third group (P) of Piedmontese breed animals (Bos taurus) was included to serve as a comparative model for the level of meat tenderness. The procedures were efficient and preserved the metal–protein structure, enabling calcium detection in protein spots by SR-XRF at a given molecular weight range of 14–97 kDa. Two proteins (pyruvate kinase and albumin) were inferred to be related to the phenotypical differences in animals from the different groups.

Introduction

The application of knowledge of the muscle proteome is a new tool in meat science. Several studies have been published in recent years with proteomic data that described, among other things, storage and maturation processes and responses to different conditions of meat processing (Hollung and Veiseth-Kent, 2012, Jia et al., 2006, Lonergan et al., 2010). Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) was used in most of those studies in combination with mass spectrometry (MS) to identify specific proteins.

The identification of differences in protein expression and/or the assessment of new or absent proteins (and, thus, possible biomarkers) in proteomics studies is one of the main applications of 2D-PAGE (Bandow et al., 2008). Proteins separated by 2D-PAGE can then be identified by their unique characteristics measured by MS. The MS is an indispensable analytical tool in chemistry, biochemistry and related biological sciences. Among other purposes, MS is used for the analysis of combination libraries and cells and the sequencing of biomolecules in tissues (Gross, 2011).

The greatest challenge of improving the quality of meat products using proteomics involves understanding which variations affect the tenderness of meat-producing species. In this context, changes in energy metabolism in the bovine longissimus thoracis muscle proteome were evaluated by Jia et al. (2006) during the pre- and post-slaughter (ante mortem and post mortem) periods. The samples of this study were collected 4 days before slaughter and 60 min after slaughter. The results suggest that 24 proteins involved in the glycolytic pathway and/or heat-shock proteins (HSP) showed significant changes in expression between the periods assessed.

Lametsch, Roepstorff, Moller, and Bendixen (2004) used SDS–PAGE and 2D-PAGE methods combined with MS to obtain an understanding of myofibrillar protein degradation during the post mortem period. These authors found that the μ-calpain isoform is responsible for the degradation of actin, the myosin heavy chain (MHC), the myosin light chain I, tropomyosin α4, tropomyosin α1, capZ, pyruvate kinase and thioredoxin proteins. The authors classify actin and MHC as the main μ-calpain substrates because they affect meat tenderness when they undergo less degradation.

Conversely, in a study by Kim et al. (2008), while alpha-actinin expression was found to be higher in animals submitted to the standard of quality A (tender meat with high marbling), the expression of T-complex proteins, heat shock protein 27 (HSP27) and inositol 1,4,5-triphosphate receptor type 1 (IP3R1) was found to be higher in the B-standard animals (tough meat without marbling). Kim et al. (2008) conducted their study with the goal of testing the use of these proteins as biomarkers for meat tenderness and marbling and to determine if the quantitative expression of HSP27 and IP3R1 was correlated with marbling, tenderness and free calcium levels; the authors found that these proteins could indeed be used as biomarkers of Hanwoo beef quality.

However, few studies have been performed on animals of the Bos indicus genotype. Furthermore, other studies usually seek to identify environmental factors or genetic groups associated with meat tenderness. A study aimed at identifying animals within a given breed expressing genetic potential for meat production with economically important organoleptic characteristics (including tenderness) is innovative and worthwhile. The present study aimed to perform the separation of longissimus thoracis (LT) muscle proteins by 2D-PAGE, to identify calcium ions in protein spots by synchrotron radiation X-ray fluorescence (SR-XRF) and to characterise proteins by electrospray ionisation mass spectrometry (ESI-MS) from Nellore breed (B. indicus) cattle with different grades of meat tenderness.

Section snippets

Animals and sample collection

All procedures with animals were conducted from the ethical research standards, established by the São Paulo State University. One-thousand-eighty-four (1084) bulls of the Nellore breed, aged 20–24 months, were used in this experiment. Performance records and the pedigree of the animals were provided by the Delta Gen, CRV Paint and Nelore Qualitas® breeding programs. Animals destined for slaughter were divided into several contemporary groups, which were formed mainly by year of birth and animal

Beef quality

The model detected differences in the grade of the animal meat tenderness among the phenotypical classes (experimental groups). The animals selected to form the TE group (tender meat) had SF values ranging from 2.40 to 4.70 kg, while the animals selected for the TO group (tough meat) had SF values ranging from 5.30 to 10.11 kg. Therefore, the samples of P group had average SF 2.76 kg. All this results about meat quality analyses of experimental groups can be observed at Table 1, which is evident

Conclusions

The techniques used in this study (2D-PAGE) efficiently separated the proteins found in LT muscle tissue samples from groups of animals with different grades of meat tenderness in this metalloproteomics study of Nellore cattle. The correlations found in the gel replicates indicated that the total protein extraction procedures were efficient and preserved the metal–protein structure, enabling calcium detection in protein spots by SR-XRF at a given molecular weight range of 14–97 kDa. Two proteins

Acknowledgements

The authors thank the São Paulo Research Foundation (Fundação de Amparo à Pesquisa do Estado de São Paulo, FAPESP, Processes 2009/16118-5, 2010/51332-5 and 2011/14850-0), the Government Funding Agency (Coordenadoria de Aperfeiçoamento de Pessoal de Nível Superior – CAPES) and the Institute of Biosciences Foundation (FUNDIBIO) for the funding.

References (30)

  • A. Rodas-González et al.

    Establishing tenderness thresholds of Venezuelan beef steaks using consumer and trained sensory panels

    Meat Science

    (2009)
  • F.M. Verbi et al.

    Metal-binding proteins scanning and determination by combing gel electrophoresis, synchrotron radiation X-ray fluorescence and atomic spectrometry

    Journal of Biochemical and Biophysical Methods

    (2005)
  • S. Wang et al.

    Lipolysis and the integrated physiology of lipid energy metabolism

    Molecular Genetics and Metabolism

    (2008)
  • J.E. Bandow et al.

    Improved image analysis workflow for 2-D gels enables large-scale 2D gel-based proteomics studies – COPD biomarker discovery study

    Proteomics

    (2008)
  • E.G. Bligh et al.

    A rapid method of total lipid extraction and purification

    Canadian Journal of Biochemistry Physiology

    (1959)
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      The abundance of HSPA1A corroborates the results of Bjarnadóttir et al. [17] and Carvalho et al. [9] who observed lower abundance of this protein in tender meat samples from the LT muscle of Norwegian Red and Nellore cattle, respectively. The protein abundance result of HSPA1B obtained in the present study agrees with the study of Baldassini et al. [6] in which abundance of this protein was lower in tender meat samples of Nellore cattle. On the other hand, proteins HSPA1A and HSPA1B identified in meat of Maremmana (Bos taurus) cattle were up-regulated in tender meat samples [4].

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