Targeted lipidomics: Planned discovery

Lipidomics Gateway (22 July 2009) [doi:10.1038/lipidmaps.2009.16]

A targeted lipidomic strategy allows rapid and sensitive identification of a novel family of mammalian acyl amino acids.

Selected representations of acyl amino acids from the LIPID MAPS structure database: 1. N-arachidonoyl tyrosine 2. N-docosanoyl taurine 3. N-oleoyl glycine 4. N-arachidonoyl glycine 5. N-palmitoyl glycine

To realize the goal of understanding how lipids function in health and disease, lipidomics approaches must identify and quantify all of the lipid species in a biological system, and how their levels change under different circumstances. Preferred techniques include separation of extracted lipids by high performance liquid chromatography (HPLC), coupled with mass spectrometric (MS) analysis. Identification of novel compounds using these approaches can be time consuming, limited to a few species, or lacking in the sensitivity to detect low abundance molecules such as signaling lipids. To overcome these limitations in the identification of a predicted family of novel, low-abundance acyl amino acids, Bo Tan et al. adapted the approach using ideas from proteomics. Reporting in the Journal of Lipid Research, the authors have identified 50 acyl amino acids from rat brain extracts and suggest that their techniques will expedite the characterization of these and other novel low-abundance signaling lipids ¹ .

Acyl amino acids consist of a fatty acid conjugated with an amino acid, and are related to other fatty acid amides including anandamide and oleoyl ethanolamine. Several synthetic acyl amino acids have been reported to have psychopharmacological effects but only a few endogenous species have been identified in mammals, including N-arachidonoyl glycine (NAGly). There are over a hundred possible combinations of amino acids with endogenous fatty acids which, combined with those already found in low abundance, suggested to the authors that a large family of these molecules might exist in mammals. Predicting the existence of these compounds helped the authors to identify them from MS data, and enabled the synthesis of a set of standards for comparative analysis. The group designed an acyl amino acid chemistry based purification procedure that used solid phase extraction columns to enrich the target lipids. This reduced the initial sample of methanol-extracted rat brain lipids to a volume small enough for the use of nano-HPLC, which is more sensitive than standard HPLC, coupled with tandem mass spectrometry.

In the first round of tandem MS, singly-charged molecular ions are detected and target ions selected for fragmentation and further analysis. Manual selection decreases efficiency by restricting the number of compounds that can be identified in a single run. Therefore the group used information-dependent acquisition (IDA) — a computer-aided selection process used in proteomics research — that generated over a thousand spectra of potential molecules of interest per run. The authors were able to rapidly analyze these data by creating a program that searches for the mass of theoretical acyl amino acids and their most prominent fragment ions ² . The potential endogenous acyl amino acids identified were then confirmed by comparing their spectra with a database compiled from the synthetic standards.

The high sensitivity of nano-HPLC, combined with the efficiency of the detection protocol, enabled the identification of 50 novel endogenous acyl amino acids in a relatively short time, and confirmed the authors' prediction that a large family of these molecules exists in mammals. Most of the rat brain acyl amino acids identified were quantified against a deuterated standard but further work is needed to define the levels of these compounds in different tissues and whether, similar to NAGly, they have any signaling functions. Adapting this approach to characterize other lipid families might lead to the discovery of other novel low-abundance lipids — researchers just need to know what they expect to find.

Emma Leah