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 subproteome of Longissimus thoracis at 24 hours 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. 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.

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.

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.