Skeletal muscle-specific metabolic labeling of nascent proteome in vivo

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Effective organ crosstalk is essential for maintaining systemic homeostasis, with skeletal muscle serving as a key regulator of metabolism. Beyond its primary functions in force generation and movement, skeletal muscle synthesizes and secretes proteins that influence local and distal biological processes. While the muscle proteome has been extensively studied, a comprehensive, unbiased investigation of its newly synthesized proteins, remains limited. A major challenge is distinguishing proteins synthesized in the skeletal muscle from those derived from circulation or neighboring tissues. To address this challenge, we utilize a well-established mutant MetRS-based metabolic labeling method that selectively incorporates azidonorleucine (ANL) into newly synthesized proteins within the skeletal muscle. Combined with click chemistry-based proteomics, this method offers a powerful tool to investigate the nascent muscle proteome. Here, we apply this approach to study proteomic remodeling in response to endurance exercise and aging. By labeling newly synthesized proteins in vivo across exercised and sedentary cohorts, as well as young and aged mice, we identify the dynamic changes in muscle protein synthesis that drive its structural and functional plasticity. This strategy has the potential to enhance our understanding of how the muscle proteome and secretome regulate inter-tissue communication as part of the adaptive response to physiological challenges.

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