Shotgun Analysis of Diacylglycerols Enabled by Thiol-ene Click Chemistry

Diacylglycerols (DAGs) are a subclass of neutral lipids actively involved in cell signaling and metabolism. Alteration in DAG metabolism has been associated with onset and progression of several human-related diseases. The structural diversity of DAGs and their low concentrations in biological samples call for the development of methods that are capable of sensitive identification and quantitation of each DAG species as well as rapid profiling when a biochemical pathway is perturbed. In this work, the thiol-ene click chemistry has been employed to introduce a charge-tag, namely, cysteamine (CA), at a carbon-carbon double bond (C═C) of unsaturated DAGs. This one-pot photochemical derivatization is fast (within 1 min), universal (monotagging) for DAGs varying in fatty acyl chain lengths and the number of C═Cs, and suitable for small sample volume (e.g., 1-50 μL plasma). Because of the presence of the amine group in CA, tagged DAGs showed at least 10 times increase in response to electrospray ionization as compared to conventional ammonium adduct formation. Low-energy collision-induced dissociation of CA tagged DAGs allowed confident assignment of fatty acyl composition. A neutral loss scan based on characteristic 95 Da loss (a combined loss of CA and H₂O) of tagged DAGs has been established as a sensitive means for unsaturated DAG detection (limit of detection = 100 pM) and quantitation from mixtures. The analytical utility of CA tagging was demonstrated by shotgun analysis of unsaturated DAGs in human plasma, including samples from type 2 diabetes mellitus patients.