The phospholipase-catalyzed release of free fatty acids (FFAs) from phospholipids is implicated in many critical biological processes such as neurotransmission, inflammation, and cancer. However, determining the individual change in FFAs generated during these processes has remained challenging due to the limitations of current methods, and has hampered our understanding of these key mediators. Here, we developed an "iTRAQ"-like method for profiling FFAs by stable isotope tagging (FFAST), based on the differential labeling of the carboxyl group and designed to resolve analytical variance, through a multiplexed assay in cells and subcellular fractions. With nanomolar sensitivity, this method revealed a spectrum of saturated FFAs elicited during stimulation of exocytosis that was identical in neurons and neurosecretory cells. Purified secretory vesicles also generated these FFAs when challenged with cytosol. Our multiplex method will be invaluable to assess the range of FFAs generated in other physiological and pathological settings.
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| http://dx.doi.org/10.1016/j.chembiol.2015.09.010 | DOI Listing |
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