Diacylglycerols (DAG) have a central role in early receptor-mediated signal transduction events for many types of cells. These lipid molecules also appear to be involved in cell differentiation, cell growth and apoptosis. Previously existing assays for this class of molecules are sensitive only in the picomole range, if total DAG levels are examined, or require significantly higher levels if one is interested in a molecular species profile. There are many questions regarding the relative role of DAG species as well as questions about when and where they are generated. This application seeks to refine a new technique to measure the generation of diradylglycerols, acyl or alkyl linked. This technique provides 1000 fold better sensitivity than conventional methods, molecular species specificity and mass measurements of these species. The method employs gas chromatography/negative ion chemical ionization mass spectrometry (GC/NICIMS) to separate and measure selected molecular species of DAG using internal deuterated standards for quantitation. The application seeks to fully develop this approach so that both molecular species of DAG and phospholipids may be analyzed with high sensitivity. In addition, a new method to profile phospholipid species, without a prior knowledge of the species present is proposed. When this latter method is fully developed, it may be possible to predict which DAG species deserve attention using our other proposed methodologies. This latter technique should allow investigators to understand the extent to which cells selectively generate specific molecular species. We naturally expect that having a highly sensitive assay for DAGs will facilitate studies of this important second messenger in a wide variety of cells systems and cell organelles.
