234. Low-Ionic-Strength Proteomic Profiles Reveal Structural, Membrane, and Metabolic Features of Divergent Aged Pork Eating Quality.

Abstract Advances in proteomic instrumentation and data analysis now enable larger, more informative experiments. Although molecular drivers of fresh pork quality are better understood, we hypothesize that proteomic phenotypes also explain variation in aged pork eating quality. A subset of 98 pork loins was selected for the proteomic experiment from a larger population of 361 loins. The selection criteria for proteomics was based on quartile rankings of trained sensory tenderness and juiciness of loins after 14 d aging, where “POOR” loins (n = 45) ranked in the 1st and 2nd quartile and “BEST” loins (n = 53) ranked in the 3rd and 4th quartile for both trained sensory tenderness and juiciness. The meat quality phenotypes were compared but are not the primary focus of the current study. BEST chops had higher (P < 0.05) pH, greater tenderness and juiciness, and lower purge and cook loss than POOR, while lipid content did not differ. Proteins soluble in a low-ionic-strength buffer (40 mM Tris-HCl, pH 8.5; 1 mM EDTA) were extracted, clarified by centrifugation, and standardized to 10 mg/mL. A pooled sample (equal volumes of all samples) was prepared for quality control. Extracts were reduced, alkylated, trypsin-digested, and peptides (200 ng/µL) were spiked with PRTC internal standard (25 fmol/µL; Thermo Scientific). Samples were analyzed by data-independent acquisition on an Orbitrap Astral coupled to a Vanquish Neo UHPLC (Thermo Scientific). Raw files were processed in Proteome Discoverer (v3.2) with CHIMERYS against the Sus scrofa UniProt reference proteome FASTA file. Differential abundance was evaluated using moderated t-tests (limma; R v4.4.2) with Benjamini–Hochberg adjusted P < 0.05. Criteria for significance included adjusted P value < 0.05 and |log2FC| >0.50. In total, 1,231 proteins (present in > 50% of samples; >1 unique peptide) were retained; 120 were more abundant in BEST and 48 in POOR. BEST samples showed a greater abundance of structural/cytoskeletal proteins (e.g., desmin, nexilin, synemin, filamin, titin), consistent with proteolysis and release into the low-ionic-strength fraction. Markers of membrane disruption extended to internal compartments, particularly the sarcoplasmic reticulum; higher sarcalumenin and calsequestrin provided evidence of organelle membrane disruption in BEST pork. AMP deaminase was more abundant in BEST, suggesting diversion of AMP away from glycolysis activation and attenuation of postmortem pH decline. Lower creatine kinase and adenylate kinase in BEST further supported upstream differences in energy metabolism associated with pH. Overall, the proteomic data support a coherent biological explanation for divergence between BEST and POOR pork in which structural remodeling, membrane/organelle instability, and metabolic regulation are intertwined features of postmortem processes linked to pH. These findings provide a biological framework for understanding divergent aged pork eating quality and support continued evaluation of low-ionic-strength soluble proteins as markers of high-quality pork.