TMT-Based Proteomic Profiling of Serum Reveals the Impact of Developmental Stage and Generation in Beef Cattle

存明 杨^1,2*Zhen Ma^1,2Xiao Wang²Yaseen Ullah¹Kexin Xu^1,2Mingrui Fan^1,2Xiangmin Yan^2*Zhao Zongsheng^1*Lei Chen^1*

• ¹Shihezi University College of Animal Science and Technology, Shihezi, China
• ²Xinjiang Academy of Animal Science Institute of Animal Husbandry, Urumqi, China

Abstract: Xinjiang Brown cattle are an important beef breed in Northwest China. Although multigenerational selective breeding has improved their growth performance, the accompanying molecular adaptations and potential physiological trade-offs remain insufficiently elucidated at the systemic level. This study aimed to decipher the dynamic serum proteomic profiles shaped by both ontogeny and generational selection in Xinjiang Brown cattle, and to identify the associated key proteins and pathways. Serum samples from 18 bulls across three generations (A, B, C) at 3 and 9 months of age were analyzed using Tandem Mass Tag (TMT)-based quantitative proteomics. Under stringent quality control (FDR < 1%), 583 high-confidence proteins were identified. Differentially expressed proteins (DEPs) were screened using thresholds of |fold change| ≥ 1.2 and p-value < 0.05. Bioinformatic analysis was performed to elucidate protein functions, and 12 key DEPs were validated by Parallel Reaction Monitoring (PRM). Serum proteome variations were primarily driven along two dimensions: age and generation. Age-related differences, marked by upregulated proteins such as APOA4 at 3 months, were enriched in lipid metabolism and skeletal development pathways. In contrast, generational shifts revealed a coordinated downregulation of proteins involved in immune-vascular homeostasis, including VWF, THBS1, TGFB1, CCN2, and TIMP3, alongside an increase in the chromatin regulator HIST2H2AC. Enrichment analysis highlighted "platelet activation" and the "TGF-beta signaling pathway" as significantly altered intergenerational regulatory networks. PRM validation confirmed the reliability of the proteomic data. This study reveals that the breeding strategy for Xinjiang Brown cattle prioritizes shaping a proteomic landscape that promotes growth and metabolism, potentially at the cost of attenuated immune-vascular reactivity. The identified panel of candidate proteins provides a molecular framework for evaluating breeding outcomes and designing balanced selection strategies. Follow-up research should further investigate the functions of these candidate proteins and validate their predictive value for health and production performance in independent herds.