Measuring C2C12 Myotube Protein Expression Changes in Inflammation using Punch-P

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.