Proteomic characterization of dark-cutting beef longissimus lumborum across varying visual severities

Dark-cutting beef, characterized by high ultimate pH and decreased surface redness, presents a significant quality issue and economic loss. This study investigated proteomic alterations across varying severities of dark-cutting beef using a combination of compartmental protein extraction and Tandem Mass Tag (TMT) labeling. Longissimus lumborum muscles from four beef categories (normal, shady, moderate, and moderately severe) were analyzed for measuring the different forms of myoglobin (deoxy- and oxy-myoglobin) and protein expression with high-resolution mass spectrometry (n = 6/treatment). Proteins were extracted into cytoplasmic/cytoskeleton (fraction 1) and nuclear/membrane (fraction 2) compartments. Moderately severe and moderate dark-cutting steaks exhibited significantly (P < 0.05) lower concentration of oxymyoglobin and higher concentrations (P < 0.05) of deoxymyoglobin at 60 min when compared with normal-pH steaks. In fraction 1, 2,144 proteins were identified, with 967 and 98 differentially abundant proteins (DAPs) in moderately severe and moderate samples, respectively. In fraction 2, 3,449 proteins were detected, of which 1,439 were differentially abundant in moderately severe samples (724 upregulated and 715 downregulated). Major metabolic changes included reduced abundance of glycogen metabolism and ribosomal proteins, along with increased expression of proteins related to gluconeogenesis, oxidative phosphorylation, and pentose phosphate pathway. At higher severities (pH ≥6.4), stress-related responses such as (hypoxia-inducible factor 1) HIF-1-signaling, endoplasmic reticulum stress, and ubiquitin-proteasome activity were more prominent, indicating disruption in tissue homeostasis. This is the first study to apply compartmental extraction with TMT-based proteomics to characterize subcellular proteome changes in dark-cutting beef, offering new insights into molecular pathways linked to condition severity.