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Lipid analysis: Click, stick... and release

Lipidomics Gateway (24 February 2010) [doi:10.1038/lipidmaps.2010.4]

A new way to tag and purify individual lipid species offers detailed insights into lipid metabolism.

Reversible cobalt complexation of alkynes can be used to specifically retrieve modified lipids from complex cellular mixtures.

Powerful mass spectrometry (MS) techniques have revolutionized lipid research and spawned the field of lipidomics, but even these methods are thwarted by the complexities of lipid metabolism. Many distinct but related cellular lipids generate identical signatures in MS data – they are isobaric – so important metabolic changes remain challenging to detect. Borrowing from the principles of click chemistry, Alex Brown and colleagues now demonstrate a new way to reversibly tag and isolate lipid species. Their method, reported in Nature Chemical Biology, allows for the selective purification of lipid metabolites and their binding partners, and promises unprecedented insights into lipid signaling and oxidation pathways.

Labeling might seem to be an obvious tactic for following cellular lipid metabolism, but there are several challenges. Radioactive labels do not alter lipid processing and can be used to monitor changes within some lipid classes, but the problem of isobaric natural species still masks many subtle alterations. Bulkier labels might interfere with cellular processes, or render the lipid unquantifiable by MS because of a lack of commercially available standards for comparison.

The new technique, developed with support from the NIH-funded LIPID MAPS consortium and NIEHS, overcomes these problems by adding a tiny chemical 'handle' to lipids, which can be reversibly grabbed by complexation. Lipids of interest can be pulled from mixed samples and gently released, unchanged, for MS analysis alongside existing standards.

The authors' strategy was inspired by click chemistry, an approach for reliable chemical synthesis that exploits triple carbon–carbon – alkyne – bonds. These chemical groups are rare in naturedo not occur naturally and are generally inert to metabolismnot naturally altered, but can be deliberately modified. The authors synthesized fatty acids with a terminal alkyne modification and added them to the medium of macrophages in culture to test their uptake. Incorporation into cellular phospholipids was confirmed by a series of MS analyses, which would be too complex for routine use. Some fatty acid chains had been elongated by two carbons, indicating normal processing. Furthermore, alkyne phospholipids served as normal substrates for phospholipase D, a lipid signaling enzyme whose activity can be monitored by its use of butanol.

The alkyne bond itself does not enable the detection of lipids, but allows them to be tagged in ways that do. Classical click chemistry involves permanent modification of alkyne bonds but the authors instead used complexation with cobalt (dicobalt octacarbonyl), which can be reversed by mild oxidation. Alkyne–cobalt complexes are known to react selectively with triphenylphosphine, and a silica gel functionalized with a related compound was used to successfully capture the tagged lipids. After native lipids were washed away, treatment with ferric nitrate freed the unmodified alkynyl lipids ready for MS analysis.

This method will allow the quantification of dozens of individual lipid species of interest, away from the occluding backdrop of hundreds of other cellular lipids. Metabolites retain the alkyne bond and are detected regardless of other changes to the original molecule. Furthermore, proteins or DNA complexed with the tagged lipids should not be lost during the gentle isolation procedure and can be detected by MS analysis, revealing precise details of metabolic fate.

The work, conducted in collaboration with the labs of Ned Porter and Larry Marnett, aims to characterize the oxidative damage to membrane phospholipids caused by free radicals, and use the findings to rationally combat oxidative stress.

Related highlights:

Oct 09: Choline phospholipids: Now you see them
A new metabolic labeling technique allows in vivo imaging of choline phospholipids.

Jan 10: Here and now: The lipid revolution
Lipidomics is revolutionizing lipid research, uniting disparate themes into coherent output that will inform wider biology.

Emma Leah

- Copyright © 2010 Nature Publishing Group, a division of Macmillan Publishers Limited; used with permission


Original research paper

  1. Milne, S.B., Tallman, K.A., Serwa, R., Rouzer, C.A., Armstrong, M.D. et al. Capture and release of alkyne-derivatized glycerophospholipids using cobalt chemistry.

    Nat. Chem. Biol. 6, 205-207 (2010). doi:10.1038/nchembio.311

Further reading

  1. Kolb, C.H., Finn, M.G. & Sharpless, K.B. Click chemistry: diverse chemical function from a few good reactions.

    Angew. Chem. Int. Ed. 40, 2004-2021 (2001). doi:10.1002/1521-3773(20010601)40:11<2004::AID-ANIE2004>3.0.CO;2-5

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