Optimization of a fluorogenic assay to determine caspase 3/7 activity in meat extracts.

Usefulness of general-purpose fluorogenic assay kits to determine caspase 3/7 activity of biological extracts is highly compromised in meat-based samples due to their scarce enzyme concentration. In the present work, a straightforward protocol is presented with two main purposes: 1) to enhance sensitivity of the fluorogenic approach addressing caspase 3/7 activity in tissues showing scarce enzyme concentration such as skeletal muscle, and 2) to reduce/economize the volume of employed reagents. The enzyme extraction procedure, peptide substrate, dithiothreitol concentration and detection settings were appropriately optimized for use in microtiter-plate fluorometers. As a result, low to high enzyme activity extracts (from 10,000 to 260,000 relative fluorescence units) can be measured under developed sampling and experimental conditions. The fact that enzyme reactions took place in 96-microtiter well plates reduces the consumption of chemical compounds when analysing a high number of samples, thus contributing to environment sustainability.

Protein Biomarkers of Bovine Defective Meats at a Glance: Gel-Free Hybrid Quadrupole-Orbitrap Analysis for Rapid Screening.

An understanding of biological mechanisms that could be involved in the stress response of animal cattle prior to slaughter is critical to create effective strategies aiming at the production of high-quality meat. The sarcoplasmic proteome of directly extracted samples from normal and high ultimate pH (pHu) meat groups was studied through a straightforward gel-free strategy supported by liquid chromatography hybrid quadrupole-Orbitrap high-resolution mass spectrometry (LC-HRMS) analysis. A stepped proteomic pipeline combining rapid biomarker hunting supported by qualitative protein Mascot scores followed by targeted label-free peptide quantification revealed 26 descriptors that characterized meat groups assayed. The functional study of the proposed biomarkers suggested their relevant role in metabolic, chaperone/stress-related, muscle contractility/fiber organization, and transport activities. The efficiency, flexibility, rapidity, and easiness of the methodology proposed can positively contribute to the creation of innovative proteomic alternatives addressing meat quality assessment.

Caspase activity in post mortem muscle and its relation to cattle handling practices.

BACKGROUND: Animal handling practices are one of the factors majorly affecting animal metabolism prior to slaughter. This phenomenon increases the occurrence of meat quality defects such as dark cutting-beef, causing high economical losses in the meat industry. Under this framework, the assessment of apoptosis onset in post mortem muscle was proposed as a novel approach to reveal biochemical characteristics in several Spanish bovine breeds (Asturiana de los Valles, Retinta and Rubia Gallega) managed under different production systems (intensive versus semi-extensive) and transport/lairage conditions (mixing versus not mixing with unfamiliar animals). To do so, the activities of initiator caspase 9 and executioner caspases 3/7 were determined in Longissimus thoracis et lumborum muscle at three early post mortem times (2, 8, and 24 h). RESULTS: Breed effect and transport/lairage conditions were the most relevant factors that influenced both caspase activities over post mortem time, showing Rubia Gallega breed a completely different behavior compared to Asturiana de los Valles and Retinta breeds. Moreover, it is postulated that apoptosis cascade is initiated via the activation of caspase 9 under hypoxic or metabolic stress followed by the activation of executioner caspases 3/7. CONCLUSIONS: Assessment of apoptosis on post mortem muscle can be a novel approach to study the influence of animal handling on muscle metabolism and post mortem cell death and its consequences on meat quality traits. © 2021 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Proteomic pipeline for biomarker hunting of defective bovine meat assisted by liquid chromatography-mass spectrometry analysis and chemometrics.

A wide variety of factors prior to slaughter may affect the stress status of beef cattle, giving rise to well-known 'dark-cutting' defective meats characterised by a high ultimate pH (pHu). To understand the underlying mechanisms of pHu fluctuations in beef cattle there was studied the proteome changes caused by pre-slaughter stress through a gel-free proteomic approach. Comparative peptidomic analysis was carried out on 12 loin samples at 24 h post-mortem from Longissimus thoracis et lumborum bovine muscle of crossbred animals, previously sorted into two different groups according to their pHu values: normal (pHu < 6.0) and high (pHu ≥ 6.0). Tryptic peptides from direct protein extracts were approached by combining untargeted (intact mass, MS1) and targeted (Selected Reaction Monitoring, SRM) quantitative LC-MS assays followed by chemometric analysis. Seventeen peptide biomarkers belonging to 10 different proteins appropriately discriminated sample groups assayed. Results may promote the use of this simple and effective methodology towards the creation of new insights in meat quality research. SIGNIFICANCE: The significance of this study was the optimization of an affordable straightforward gel-free proteomic approach addressing the differentiation of the muscle sub-proteome of normal and high pHu meat samples. This strategy allowed the study of tryptic peptides from direct meat protein extracts by combining untargeted MS1 and targeted SRM quantitative assays performed by conventional LC-MS detection. Affordability, simplicity and robustness of this methodology can facilitate its readily implementation in routine protocols for quality assessment of meat.

A straightforward gel-free proteomics pipeline assisted by liquid isoelectric focusing (OFFGEL) and mass spectrometry analysis to study bovine meat proteome.

Bovine sarcoplasmic sub-proteome was studied through a straightforward gel-free pipeline supported by liquid isoelectric focusing (OFFGEL) protein fractionation coupled to liquid chromatography-mass spectrometry (LC-MS) analysis. Full-MS and data-dependent MS/MS analyses were simultaneously performed by a conventional three-dimensional ion-trap addressing targeted quantitative and untargeted qualitative research, respectively. There were unambiguously identified 47 proteins distributed along 12 OFFGEL fractions assayed. Regarding intermediate- and high-abundant peptides, bulky quantitative data processing performed by MZmine 2 freeware yielded a satisfactory linearity and coefficient of variation with r2 in the 0.95-0.99 range and about 25%, respectively. Up to 41 peptides from 20 identified proteins were relatively quantified throughout OFFGEL fractions. This reliable, flexible and affordable gel-free proteomic approach could be readily implemented by industry to improve quality assessment of protein-based food products.

Characterization of the Myofibrillar Proteome as a Way to Better Understand Differences in Bovine Meats Having Different Ultimate pH Values.

Influence of ultimate pH (pHu) on the occurrence of defective meats known as dark, firm, and dry (DFD) meats has been studied through a proteomic approach at early post-mortem times. The myofibrillar sub-proteome of longissimus thoracis et lumborum muscle from twelve loin samples from Asturiana de los Valles x Friesian yearling bulls, previously classified into two groups of six samples according to their pH values (normal, pHu < 6.0 and high, pHu ≥ 6.0), is analyzed at 24 h post-mortem. Fractionation/enrichment of muscle samples is carried out by combining OFFGEL fractionation in the pH range 4-7 followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of the retrieved liquid fractions. Four protein bands satisfactorily discriminate between meat samples with normal and high pHu. These bands are quantified by image analysis, and further identified by liquid chromatography-mass spectrometry as desmin, pyruvate kinase, myosin light chain, and myosin heavy chain-1 and -2. Coupling OFFGEL and SDS-PAGE separation with MS provides detailed and reproducible myofibrillar protein profiles enabling comparison among the sample groups assayed. This makes feasible to identify biomarkers capable to better understand pre-slaughter stress condition susceptible to give DFD meats with high pHu values.

Search for proteomic biomarkers related to bovine pre-slaughter stress using liquid isoelectric focusing (OFFGEL) and mass spectrometry.

Proteome changes derived from animals that have suffered pre-slaughter stress are a fact. In this study, Proteomic analysis was carried out on 20 bovine loin samples from Asturiana de los Valles and crossbreds cattle previously classified as normal and DFD meat at 24 h post-mortem using pH measurements. Sarcoplasmic sub-proteome of Longissimus thoracis at 24 h post-mortem was fractionated by the use of liquid isoelectric focusing (OFFGEL) in the pH range 3-10, followed by SDS-PAGE analysis of each retrieved fraction. The protein fractionation profile showed high reproducibility along the different sample groups. Five protein bands showed significant differences (p < 0.05) between the two groups, allowing discrimination between them. Proteins present in these bands, which were identified by LC-MS, were actin, phosphoglucomutase-1, alpha-crystallin B, heat shock protein beta-6 and heat shock protein beta-1. SIGNIFICANCE: The significance of this study relies on the optimization of OFFGEL fractionation as a promising technology to search for reliable biomarkers of pre-slaughter stress. This method separates proteins along different liquid fractions according to their isoelectric point; the obtained fractions can be further characterized by SDS-PAGE or directly identified by LC-MS. This achievement stands out as an alternative to the use of 2-DE electrophoresis in protein separation and analysis.