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.