Comparative Proteomics Analysis of Pig Muscle Exudate through Label-Free Liquid Chromatography-Mass Spectrometry

**Background:** Capital-driven animal husbandry has led to the abandonment of many local pig breeds. Autochthonous breeds like the Nero d'Abruzzo (a black domestic pig from the Abruzzo region, Italy) have potential advantages in environmental sustainability, animal welfare, and product quality. Understanding the biological differences between these local breeds and commercial hybrids is important for conservation and production improvement. Proteomics offers a high-throughput approach to identify molecular phenotypes underlying these differences.

**Methods:** Four Nero d'Abruzzo gilts and four PIC gilts at the same growth stage (live weight approximately 155 ± 5 kg) were included. All animals were reared on the same farm with similar management; Nero d'Abruzzo pigs had access to an external paddock during daylight. After a 12-hour fasting period, animals were electrically stunned and slaughtered. Diaphragm specimens were collected, and muscle exudate was obtained by centrifugation (60 min). Protein concentration was determined using the Bradford method. Equal concentrations (100 μg) of protein were purified and digested using the filter aided sample preparation (FASP) method. Nano LC-MS/MS analysis was performed using an Ultimate 3000 nanoRSLC system coupled with an Orbitrap Fusion Tribrid mass spectrometer. Protein identification was achieved using Proteome Discoverer v.2.2 with SEQUEST HT algorithm against the Sus Scrofa protein database (UniProt, downloaded April 2019). Quantitative label-free analysis was carried out using Progenesis QI for proteomics software. Differentially expressed proteins required ≥2 peptides matched, ≥1.5-fold difference in abundance, and ANOVA p ≤ 0.05. Three proteins (catalase, glutathione peroxidase, fatty acid synthase) were confirmed by Western blot using specific antibodies, with three technical replicates per experiment. PANTHER and STRING databases were used for functional classification and protein-protein interaction analysis.

**Key Results:** The proteomic approach identified 7488 peptides belonging to 1496 proteins from Nero d'Abruzzo muscle exudate and 6169 peptides belonging to 1283 proteins from PIC muscle exudate, with 1669 unique proteins identified in total. A total of 100 proteins changed significantly in abundance between breeds: 83 were upregulated in Nero d'Abruzzo and 17 were upregulated in PIC. Nine proteins upregulated in Nero d'Abruzzo showed a fold change higher than 10, including fatty acid synthase (fold change: 16.62). Seventeen showed a fold change between 4 and 9, including glutathione peroxidase (fold change: 5.31) and catalase (fold change: 4.06). Among PIC-upregulated proteins, three showed a fold change of about 4, and fourteen had a fold change between 1.5 and 3. PANTHER classification showed Nero d'Abruzzo-upregulated proteins were involved in cellular process (37%), metabolic process (19%), localization (14%), biological regulation (10%), and response to stimulus (9%). PIC-upregulated proteins were dominated by metabolic process (35%) and cellular process (30%). STRING analysis revealed a connected protein network of 52 nodes with a PPI enrichment p-value of <1.0 × 10^-16 (20 expected edges vs. 139 detected edges). Western blot confirmed that catalase, glutathione peroxidase, and fatty acid synthase (both 25 kDa and 70 kDa bands) were significantly upregulated in Nero d'Abruzzo compared to PIC (p < 0.05).

**Clinical Implications:** This study provides the first comprehensive proteomic characterization of muscle exudate from the Nero d'Abruzzo pig breed compared to commercial hybrids. The upregulation of fatty acid synthase in Nero d'Abruzzo aligns with the known tendency of rustic breeds to accumulate intramuscular lipids, which may influence meat quality traits. The increased expression of antioxidant enzymes catalase and glutathione peroxidase suggests that outdoor rearing systems enhance the antioxidant defense capacity of these animals, potentially improving their health and stress resilience. These findings identify candidate proteins that could serve as molecular markers for breed-specific production performance and may inform breeding programs aimed at preserving local genetic resources while improving meat quality and animal welfare.