Molecular analysis of tenderstretch on postmortem bovine skeletal muscle protein composition using 2-dimensional electrophoresis based proteomics

Using 2-dimensional electrophoresis based proteomics we have investigated the protein expression profile of bovine M. longissimus thoracis et lumborum at day 2 (n=3) and day 14 (n=3) in tenderstretched and conventionally hung carcasses. From 1248 protein features detected by image analysis, 191 manually curated spots were subjected to statistical analysis. From this initial analysis, at day 2 postmortem; 33 spots were up regulated and 10 were down regulated (P.<0.01); whilst at day 14 postmortem, 60 up regulated and 20 down regulated protein spots (P.<0.01) were found in the tenderstretched samples. Mass spectrometry (MALDI TOF-TOF and LTQ linear ion trap) will be used to identify some of these proteins and it is anticipated that this will lead to a greater understanding of the molecular mechanisms involved in tenderisation. Introduction ‘Tenderstretch’ has been shown to help prevent sarcomere shortening in postmortem skeletal muscle and subsequently decrease shear force and improve meat tenderness in some commercially important musclesstriploin, topside, silverside and rump (Troy, 1999). Tenderstretch has also been reported to reduce variability in palatability scores in beef by up to 25% compared to conventionally hung samples (Sorheim et al., 2001; Thompson, 2002). However, little is known about the molecular mechanisms involved in the stretching of muscle postmortem. It has been suggested that ‘tenderstretch’ may affect the structural integrity of the sarcomere by aiding in the degradation of the Z-disk (Hopkins and Thompson, 2001). As well as the physical tearing of muscle fibres due to increased stretch in the muscle postmortem, other theories regarding the molecular contribution of ‘tenderstretch’ to increased meat tenderness include an increased rate of proteolysis due to the stretch induced exposure of potential proteolytic substrates within the muscle fibre (Hwang et al., 2003) and a possible activation of calcium dependant proteases due to an elevation of intracellular Ca levels caused by the stretching of the muscle (Armstrong et al., 1993). Genetic studies have indicated that pre-rigor stretching causes a decline in both additive and phenotypic variance in shear force, while heritabilities were not changed, genotype differences were reduced. This suggests that more severe stretching of muscle pre-rigor may minimise genotype effects on tenderness (Burrow et al., 2006). Materials and methods Crossbred heifers (n=3) were captive bolt stunned and exanguinated conventionally at the Meat Industrial Development Unit, Ashtown Food Research Centre, Dublin 15. The carcasses were electrically stimulated at 60V for 20 seconds. Half of each carcasses (n=3) was conventionally hung while the other half (n=3) was ‘tenderstretch’ hung by the aitch bone. M. longissimus thoracis et lumborum samples were taken from the excised muscle for protein analysis at day 2 and day 14 postmortem. Samples were snap frozen in liquid nitrogen and stored at -80oC until required. Both sarcomere length (day 7 postmortem) and Warner Bratzler shear force (day 14 postmortem) were measured according to the methods of Cross et al. (1980) and Shackelford et al. (1991). Myofibrillar protein extraction: Approximately 110mg of powdered tissue was homogenised in 1ml lysis buffer containing 7M urea, 2M thiourea, 2% CHAPS, 1% DTT, and 0.8% Pharmalyte pH 4-7 nonlinear according to the method of Hwang et al. (2005) with minor modifications. Protein Quantification: The concentration of protein present in the samples was determined using a modified Bradford assay according to the method of Ramagli and Rodriguez (1985) using bovine serum albumin (BSA) in lysis buffer as standard reference. 2-dimensional electrophoresis: 100ug of solubilised myofibrillar protein was loaded onto a ImmobilineTM DryStripTM, 24cm, pH 4-7 using an in-gel rehydration method as described by Rabilloud et al. (1994) and Sanchez et al. (1997). The first and second dimensions were carried out according to the protocol detailed in Focking et al. (2006). Iso-electric focussing (IEF) was carried out for 75kVh at 20oC (Ettan