The Lipid Library

LIPIDOMICS

It was perhaps inevitable that the sciences of genomics, proteomics, metabolomics, glycomics and so forth would lead to the ‘new’ science of lipidomics. The first mention of this that I could find in the literature was in a paper in 2001, which referred to the ‘lipidome’, i.e. the complete spectrum of lipids in a tissue or organelle. From 2002 onwards publications using the term lipidomics have appeared in increasing numbers. Lipids have been the Cinderellas of the biological world for far too long, and it is refreshing to see the sudden burst of interest in these fascinating molecules and in the techniques for their analysis.

In preparing the documents for the section of this website dealing with lipid composition and biology, I have become aware that every single lipid class has a highly specific function that is independent of its role as a source of energy or as a building block of membranes. Lipids are relatively small hydrophobic molecules, which are good candidates for signalling purposes. The fatty acid constituents have well-defined structural features, such as cis-double bonds in particular positions, which can carry information by binding selectively to specific receptors. Phospholipids have hydrophilic sites that can bind via hydrogen bonding to membrane proteins and influence their activities. Glycolipids carry complex carbohydrate moieties that have a part to play in the immune system, for example. Through their various biological activities, lipids have been implicated in a number of human disease states both in detrimental and beneficial ways. Every scientist should now be aware that lipids are just as interesting as all the other groups of organic compound that make up living systems.

The aim of lipidomics is to relate lipid compositions of biological systems to their physical properties and biology. A brief definition might be –

The analysis of lipids on the systems-level scale together with their interacting factors.

Alternatively, the more comprehensive definition [1] may be preferred –

The full characterization of lipid molecular species and of their biological roles with respect to expression of proteins involved in lipid metabolism and function, including gene regulation.

Both definitions contain two main elements. In relation to the analysis of lipids they require a full determination of all the lipids present and the nature of the molecular species of each lipid class in the biological sample being studied. Secondly, they suggest that such analytical data must be related to the biological function of lipids through knowledge of such enzymes, genes and other factors that may relevant. It is also implied that the metabolic and physical relationships of lipids to enzymes, receptors and non-lipid signalling molecules in their membrane environments must be considered. The aim is eventually to integrate all the various ‘omics’ into a single framework of cellular metabolism.

There is a danger that ‘lipidomics’ becomes simply the latest ‘buzz word’ to describe every study involving analyses of molecular species, even of a single lipid class in a tissue. I hope editors of journals will be sensitive to misuse of the term.

Is the subject really a new one? To answer this question, I have selected two papers from the 1960s that seem to me to fully satisfy the above definitions of lipidomics, at least in terms of the general knowledge of biochemistry of their time [2,3]. The main lipid classes in rat liver were isolated and quantified, the molecular species of each were analysed, and the stereospecific positional distributions of fatty acids in each lipid class were determined with high precision. Finally, the results were interpreted fully in the light of existing knowledge of biochemical pathways. Of course, I could have chosen many other papers from this era.

If the subject is not new, the methodology that is now being applied to the analysis of lipids in the name of lipidomics certainly is novel and extremely powerful. Modern mass spectrometry methods involving ionization by electrospray (ESI), fast atom bombardment (FAB), atmospheric pressure chemical-ionization (APCI), atmospheric pressure photo-ionization (APPI), and matrix-assisted laser desorption (MALDI) techniques are highly sensitive and can produce excellent quantitative data. There may be no need for extensive sample preparation or for a chromatographic interface with the instrument, since samples can be injected directly. In addition, tandem mass spectrometry methods greatly enhance the information obtainable. While the instruments are costly, they are becoming more affordable.

We have no single method for the analysis of a complex mixture of lipid classes that does not have limitations, and arguably direct-inlet mass spectrometry has fewer than most. However, are there drawbacks to this methodology of which we should be aware? I retired from active research before the new mass spectrometry techniques became available, so my knowledge of the subject is derived from my reading of the literature not from personal experience. Please consider the comments that follow in this light.

Mass spectrometry of intact lipids is still a highly specialised field that needs skilled and knowledgeable operators. A vast amount of data can be produced over a relatively short period, so time that otherwise would have been spent on derivatization and chromatography is now spent analysing this information on a computer. Until recently, newcomers to the field were obliged to develop their own software to handle these data, but happily, software tools are now being made available free of charge that enable high-throughput analysis of extensive data sets from lipid extracts [4]. Information is available on positional distributions of fatty acids in glycerophospholipids by modern mass spectrometry methods, but not with the precision of classical methods. The fatty acids in the primary and secondary positions in triacyl-sn-glycerols can be distinguished by mass spectrometry, but full stereospecific analysis is not possible, for example. Indeed, all chiral lipids may require a chromatographic step for definitive characterization. Then, there is the problem of lipids that have the same molecular weights, and a recent paper highlights the difficulty of differentiating phosphatidylglucose and phosphatidylinositol by mass spectrometry, suggesting the former might easily be missed in samples [5]. The same problem arises with phosphatidylglycerol and lysobisphosphatidic acid. There must be many more such examples. Indeed, the use of the term ‘shotgun lipidomics’, which is increasingly being used, appears to recognise the limitations of this kind [6].

The value of interfacing the mass spectrometer to a liquid chromatography system has been highlighted in a recent review of methodology for the analysis of sphingolipids [7]. The authors state “liquid chromatography tandem mass spectrometry (LC MS/MS) is currently the only technology with the requisite structural specificity, sensitivity, quantitative precision, and relatively high-throughput capabilities for such analyses in small samples (~10⁶ cells)”. I am sure that this view is correct and that chromatographic separations will continue to have great importance for the foreseeable future.

While mass spectrometry alone can give useful information on fatty acid compositions, the precision and robustness of the flame ionization detector will ensure that gas chromatographic analysis will continue to be the method of choice for detailed fatty acid analysis. Mass spectrometry will certainly remain an invaluable aid to identification. The enzymic methodology for determination of positional distributions of fatty acids in glycerolipids, which has now been available for nearly fifty years, will still be required for accurate analysis, especially in triacyl-sn-glycerols. Nuclear magnetic resonance spectroscopy will continue to have its place, as will the various optical spectroscopy methods. In short, regardless of the power of the newer mass spectrometry methods, I am encouraged to believe that the science of ‘lipidomics’ is going to continue to need skilled analysts and chromatographers for some considerable time to come.

I can recommend a recent book on the methodology of lipidomics [8].

References

1. Spener, F., Lagarde, M., Géloën, A. and Record, M. What is lipidomics? Eur. J. Lipid Sci. Technol., 105, 481-482 (2003).
2. Wood, R. and Harlow, R.D. Structural studies of neutral glycerides and phosphoglycerides of rat liver. Arch. Biochem. Biophys., 131, 495-501 (1969).
3. Wood, R. and Harlow, R.D. Structural analyses of rat liver phosphoglycerides. Arch. Biochem. Biophys., 135, 272-281 (1969).
4. Haimi, P., Uphoff, A., Hermansson, M and Somerharju, P. Software tools for analysis of mass spectrometric lipidome data. Anal. Chem., 78, 8324-8331 (2006).
5. Nagatsuka, Y., Tojo, H. and Hirabayashi, Y. Identification and analysis of novel glycolipids in vertebrate brains by HPLC/mass spectrometry. Methods Enzymol., 417, 155-167 (2006).
6. Han, X.L. and Gross, R.W. Shotgun lipidomics: Electrospray ionization mass spectrometric analysis and quantitation of cellular lipidomes directly from crude extracts of biological samples. Mass Spectrom. Rev., 24, 367-412 (2005).
7. Merrill, A.H., Sullards, M.C., Allegood, J.C., Kelly, S. and Wang, E. Sphingolipidomics: High-throughput, structure-specific, and quantitative analysis of sphingolipids by liquid chromatography tandem mass spectrometry. Methods, 36, 207-224 (2005).
8. Fen, L. and Prestwich, G.D. (Editors) Functional Lipidomics. (Taylor & Francis, Boca Raton) (2006).

Additional references on this topic can be found in the ‘Literature Survey’ section of the Lipid Library.

Updated: 15/9/2008 Credits/disclaimer	Scottish Crop Research Institute (and MRS Lipid Analysis Unit), Invergowrie, Dundee (DD2 5DA), Scotland	Right click on the icon to open in a new window