A Targeted, Bioinert LC–MS/MS Method for Sensitive, Comprehensive Analysis of Signaling Lipids

Signaling lipids
are key players in cellular processes. Despite
their importance, no method currently allows their comprehensive monitoring
in one analytical run. Challenges include a wide dynamic range, isomeric
and isobaric species, and unwanted interaction along the separation
path. Herein, we present a sensitive and robust targeted liquid chromatography-mass
spectrometry (LC–MS/MS) approach to overcome these challenges,
covering a broad panel of 17 different signaling lipid classes. It
involves a simple one-phase sample extraction and lipid analysis using
bioinert reversed-phase liquid chromatography coupled to targeted
mass spectrometry. The workflow shows excellent sensitivity and repeatability
in different biological matrices, enabling the sensitive and robust
monitoring of 388 lipids in a single run of only 20 min. To benchmark
our workflow, we characterized the human plasma signaling lipidome,
quantifying 307 endogenous molecular lipid species. Furthermore, we
investigated the signaling lipidome during platelet activation, identifying
numerous regulations along important lipid signaling pathways. This
highlights the potential of the presented method to investigate signaling
lipids in complex biological systems, enabling unprecedentedly comprehensive
analysis and direct insight into signaling pathways.