The proposed project is designed to advance the field of lipidomics and enable unprecedented specificity and sensitivity in profiling trace-level, bioactive lipids. A variety of fatty acid amides (oleamide, anandamide) are involved in neurological signaling pathways, and, in the case of primary fatty acid amides, are implicated in the etiology of affective disorders.
The specific aims of this project are: 1) implement high efficiency neutral lipid class separation functions (i.e., sample preparation of lipid extracts) on microfluidic devices and interface with electrospray mass spectrometry; and 2) develop specific chemical modification strategies for high efficiency labeling of neutral lipids on microfluidic devices, integrate with sample preparation functions, and interface with electrospray mass spectrometry and capillary chromatography with laser-induced fluorescence detection; and 3) measure the lipidome and profile the trace lipids of N18TG2 cells. Accomplishment of these goals will be met by a highly efficient multidimensional chromatographic system on a single microchip device for purification of neutral lipid classes prior to introduction into either a mass spectrometer or a high resolution, ultrasensitive liquid chromatograph with laser-induced fluorescence detection. The former device will allow rapid, quantitative profiling of the neutral lipids (including amides) from cell lines (e.g. N18TG2) that are known to produce amides. A modification of the device will allow the preconcentration of specific classes of lipids (e.g., primary fatty acid amides) that are known to be active at trace or ultratrace concentrations. Preconcentration by factors of up to 1000 will allow either high sensitivity MS or in-situ derivatization for ultrasensitive fluorescence detection. The latter will be used for measuring highly active lipids at sub-nanomolar concentrations and masses on the order of zeptomoles. This work will begin to answer numerous questions regarding the interactions and dynamics of bioactive fatty acid amides.
