TMT-based proteomic profiling of serum reveals the impact of developmental stage and generation in beef cattle.

Abstract

INTRODUCTION: 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- ofs 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. METHODS: Serum samples from 18 bulls across three genera- tions (A, B, C) at 3 and 9 months of age were analyzed using Tandem Mass Tag (TMT)-based quantitative proteomics. RESULTS: Under stringent quality control (FDR < 1%), 583 high-confidence proteins were identified. Diferentially expressed proteins (DEPs) were screened using thresholds of |fold change| &#x2265; 1.2 and-value < 0.05. Bioinformatic analysis was performed to elucidate protein functions, and 12 key DEPs were validated by Parallel Reaction Monitoring (PRM). Serum pro- teome variations were primarily driven along two dimensions: age and genera- tion. Age-related diferences, 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 signal- ing pathway" as significantly altered intergenerational regulatory networks. PRM validation confirmed the reliability of the proteomic data. DISCUSSION: 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 atten- uated immune-vascular reactivity. The identified panel of candidate proteins pro- vides 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.