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

Abstract

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.

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

“…Procedures for OFFGEL preparation were from Fuente‐Garcia et al. [ 16 ] with some modifications to specifically fit to the characteristics of myofibrillar sub‐proteome. Briefly, a volume containing 2 mg of total protein of each myofibrillar extract was considered for protein fractionation using an Agilent 3100 OFFGEL fractionator (Agilent Technologies, Palo Alto, CA, USA).…”

“…The 12 protein fractions obtained from each sample after OFFGEL fractionation were subjected to electrophoretic separation under reducing and denaturant conditions (SDS‐PAGE) as described previously [ 16 ] with some modifications. Electrophoresis of samples was performed in duplicate.…”

“…Chromatographic and MS conditions of analysis were from Fuente‐Garcia et al. [ 16 ] Peptide identification was done by analysis of generated MS/MS data against Uniprot KB v2017_11 (http://www.uniprot.org) and NCBIprot v20170428 protein databases using Mascot v2.3 as search engine with the following parameters: enzyme, trypsin; no fixed, or variable modifications but “Error tolerant” option enabled; mass accuracy was set to 1.2 and 0.8 Da for MS and MS/MS analyses, respectively; The option “Mammalia” was selected as taxonomy restriction parameter; decoy option was used to estimate the false positive rates by means of false discovery rate threshold from 1%. Only those tentative assignments with high individual ion scores indicating identity on extensive homology were considered as reliable results ( p < 0.05).…”

“…[ 10,14,15 ] Alternatively, we have recently proposed an innovative proteomic approach based on liquid isoelectric focusing (OFFGEL) for the specific fractionation/enrichment of the sarcoplasmic fraction hyphenated with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) and LC‐MS analysis. [ 16 ] Although it is known that sarcoplasmic proteins (stress/defense proteins and metabolic enzymes) are the ones mainly related to stress, the number of studies reporting changes occurring on myofibrillar proteome are scarce and deserves further investigation. The idea to go deep into the study of myofibrillar sub‐proteome is supported by the fact that texture properties are notably altered in DFD meats with respect to normal ones.…”

Omics Application in Animal Science—A Special Emphasis on Stress Response and Damaging Behaviour in Pigs

“…Several biomarkers have been suggested as indicators of various traits important for animal production. Studying the proteome associated with pre-slaughter stress in ruminants yielded a range of candidate proteins that underlie the conversion of muscle to dark, firm and dry muscle [ 49 , 50 , 51 , 52 ]. Specific findings in pigs, such as on heat stress [ 53 , 54 , 55 ], tail docking [ 56 ], confined housing [ 47 ], physical restraining [ 57 ], road transport [ 58 ] and inflammatory state [ 59 ] have potential to be applied to the stress response in pigs ( Figure 2 ).…”