Lipid Discovery by Combinatorial Screening and Untargeted LC-MS/MS

Abstract

We present a method for the systematic identification of picogram quantities of new lipids in total extracts of tissues and fluids. It relies on the modularity of lipid structures and applies all-ions fragmentation LC-MS/MS and Arcadiate software to recognize individual modules originating from the same lipid precursor of known or assumed structure. In this way it alleviates the need to recognize and fragment very low abundant precursors of novel molecules in complex lipid extracts. In a single analysis of rat kidney extract the method identified 58 known and discovered 74 novel endogenous endocannabinoids and endocannabinoid-related molecules, including a novel class of N-acylaspartates that inhibit Hedgehog signaling while having no impact on endocannabinoid receptors.

Figure 1. Design and validation of the lipid discovery workflow.

(A) AIF LC-MS/MS screen for molecules consisting of a polar head group H and fatty acid moiety F. A putative compound f_jh_i is identified if XIC peaks of its precursor ion [f_jh_i]⁺ (in blue) and fragment [h_i]⁺ spectra (in red) align (two-colour arrow). (B) Molecular ion and the head group fragment of N-acylethanolamines (NAE). (C) Venn diagram of endogenous NAE independently identified by all-ions fragmentation (AIF) and parallel reaction monitoring (PRM) on a Q Exactive and by multiple reaction monitoring (MRM) on a triple quadrupole mass spectrometer.

Figure 2. Identification and biological activity of N-acylaspartates.

(A) Alignment of XIC peaks of the [M+H]⁺ precursor and head group fragment (upper panel) identified NAAsp 20:4, consistently with the structure of its molecular ion (B) and independent analysis of the synthesised molecule (lower panel). (C) Shh-LIGHT2 cell reporter assay showing that NAAsp inhibit Hedgehog (Hh) signalling. Hh activation after stimulation with Smoothened agonist (SAG) or non-sterol-modified Shh was reduced by 15 μM of NAAsp 16:0; NAAsp 18:2; NAAsp 20:4; NAE 20:4 (positive control) and O-acylguanosine 18:1 (negative control) (n = 3). (D) NAAsp (10 μM) did not interact with CB₂ and very weakly with CB₁ receptors; they also did not inhibit FAAH. Ctrl stands for negative control; SR141716A (1 μM); SR144528 (1 μM) and URB597 (0.1 μM) are synthetic blockers serving as positive controls. (n = 4; ^*p < 0.05; ^**p < 0.001 and ^***p < 0.0005 vs control).