Alkyne lipids as tracers of lipid metabolism

The high complexity of cellular lipidomes and of the underlying metabolic pathways requires powerful labeling and detection systems for systematic lipid tracing experiments. Alkyne fatty acids are tracers with favorable biological properties very similar to unlabeled natural counterparts. We have developed a labeling and detection system based on alkyne lipid tracers and specialized reporter molecules that confer high specificity and sensitivity to labeled metabolites. Tracers are added to living cells and metabolites are extracted in pulse-chase setups to achieve time resolution. Copper(I)-dependent click reaction between extracted lipids and the C171 or C175 reporter molecules is followed by mass spectrometry analysis. The reporter carries a positive charge leading to improved ionization and increased sensitivity. Uniform and predictable neutral loss-type fragmentation in tandem mass spectrometry leads to reliable identification and quantification of labeled metabolites. Parallel multi-labeling with several precursors, combined with multiplexed analysis enables efficient high-content tracing. This chapter introduces the basic concepts and a step-by-step protocol with detailed explanation of key procedures to obtain optimal results.