Comparative Quantitative Profiling of Protein Lactylation Reveals a Dynamic Tissues-Specific Network Associated with Metabolic Specialization in Yaks

Protein lysine lactylation is an emerging post-translational modification with broad roles in metabolic regulation. The yak (Bos grunniens) has evolved strong metabolic adaptability on the Qinghai–Tibetan Plateau, yet its tissue-specific lactylation patterns remain poorly characterized. Here, we collected liver, muscle, and heart tissues from three adult male yaks (4.5 years; 305–355 kg) and integrated quantitative proteomics with lactylomics to map lactylation profiles across these tissues. After normalizing each lactylation site to its parent protein abundance and applying Benjamini–Hochberg correction, we identified 628, 982, and 541 differentially lactylated sites (|log2FC| ≥ 0.585, adjusted p < 0.05) in liver–muscle, liver–heart, and muscle–heart comparisons, with median fold changes of 4.11, 7.08, and 3.08, corresponding to 267, 372, and 219 proteins, respectively. Subcellular localization showed that approximately 27–30% of these sites were localized to mitochondria. Functional enrichment across these comparisons consistently highlighted pathways such as the TCA cycle (fold enrichment: 1.56–2.38) and HIF-1 signaling (up to 2.69). A total of 135 proteins were common to all three comparisons, some with both up- and downregulated sites within the same tissue pair. Our results reveal decoupling between protein abundance and lactylation levels, tissue-specific lactylation patterns on the same proteins, and expression-independent lactylation of key enzymes (e.g., LDHA, SIRT3). Functional enrichment suggests lactylation serves as a multimodal regulatory mechanism coordinating energy metabolism, protein homeostasis, and electromechanical coupling across tissues. Expression profiling of lactate-metabolizing enzymes and lactylation regulators further supports an organ-specific model of post-translational regulation. Collectively, these findings detail a complex, tissue-specific lactylation network in yaks and provide insights into the metabolic homeostasis essential for high-altitude life.