Updated project metadata. Selective identification of newly synthesized proteins provides insight on biological processes that depend on the continued synthesis of specific proteins to maintain homeostasis as well as those that are up- or down-regulated in response to changing needs. We used puromycin-associated nascent chain proteomics (PUNCH-P) to characterize new protein synthesis in C2C12 myotubes and changes resulting from exposure to a combination of lipopolysaccharide and interferon-(LPS/IFN) for either a short (4h) or prolonged (16h) time period. We identified sequences of nascent polypeptide chains belonging to a total of 1,523 proteins and report their detection within three independent samples for each growth condition. The identified proteins correspond to approximately 15% of presently known proteins in C2C12 myotubes and are enriched in specific cellular components and pathways. A subset of these identified proteins have functional characteristics and were identified only in treated samples, consistent with new protein synthesis in response to LPS/IFN. Thus, the identification of proteins from their nascent polypeptide chains provides a resource to analyze the role of newly synthesizing proteins in protein homeostasis and proteome responses in C2C12 myotubes.