The human genome encodes 11 genes belonging to the membrane bound O-acyltransferase (MBOAT) family of proteins. MBOAT family proteins are known for containing a large number of transmembrane domains and exhibiting acyltransferase enzymatic activity. While collectively these proteins cover a broad-spectrum of acylation, substrates ranging from phospholipids to peptides, each member possesses specificity with regards to the target of acylation and the lipid species utilized. Mitsugumin 56 (MG56), also known as hedgehog acyltransferase-like protein, is an MBOAT protein predominantly expressed in cardiac and skeletal muscle. MG56 resides along the terminal cisternea of the Sarcoplasmic Reticulum (SR) in striated muscle. Ablation of MG56 in mice results in seemingly normal pups at birth, however severe growth retardation is observed around day 7, shortly followed by death under starvation conditions. Skeletal muscle from knockout mice displays swollen SR and dilated vacuoles within sarcomeres, however this phenomenon is not seen in cardiac tissue. Lipid analysis of skeletal muscle via matrix-assisted laser desorption/ionization fourier transform ion cyclotron resonance mass spectrometry (MALDI FT-ICR) shows a larger abundance of phospholipids in knockout muscle compared to wild type. Interestingly, knockout muscle also shows an increased abundance of high molecular mass phospholipids, indicative of tri-acyl phospholipids. Tri-acylated phospholipids, such as N-acyl phosphatidylethanolamine (NAPE), have an additional acyl chain attached to the phosphate group, and have been shown to alter membrane size and serve as precursors to N-acyl ethanolamine (NAE), a hormone shown to be an appetite suppressant in rodents. It is our hypothesis that MG56 functions to negatively regulate NAPE production, thereby fostering normal muscle development. We are working to identify the high molecular mass lipids seen in knockout skeletal muscle in hopes of determining the substrate of MG56.