Analysis of Lipids by Mass Spectrometry

Nearly all available ionization techniques of MS have already been applied successfully to Upids, and their individual advantages and disadvantages discussed [25]. Nowadays, the overwhelming majority of lipidomic investigations makes use of electrospray ionization (ESI) MS [26], and useful protocols have been developed in this respect [27, 28]. In comparison to ESl-MS, Upid studies based on MALDl [29] or atmospheric-pressure chemical ionization (APCI) [30] are much less abun-danL and a major aim of this chapter will be to emphasize the benefits of MALDI-MS. 

FinaUy, classical electron ionization (EI)-MS is no longer a convincing method for Upid analysis due to the huge extent of fragmentation that is caused. Despite its many limitations, coupled MS-gas chromatography (GC/MS) remains the most widely established method for free acid analysis [31,32], although it is rather time-consuming and tedious. 

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Ellis, S.R., Brown, S.H., In Het Panhuis, M., Blanksby, S.J., and Mitchell, T.W. 2013. Surface analysis of lipids by mass spectrometry More than just imaging. Prog. Lipid Res., 52 329-353. 

Structural analysis of biological macromolecules and lipids by mass spectrometry... 

STRUCTURAL ANALYSIS OF BIOLOGICAL MACROMOLECULES AND LIPIDS BY MASS SPECTROMETRY 493... 

Murphy RC, Fiedler J, and Hevko J (2001) Analysis of non-volatile lipids by mass spectrometry. Chemical Reviews 101 479-526. 

Analysis of lipids by MALDI mass spectrometry has received much less attention than analysis of proteins and peptides but lipids can, nevertheless, be induced to produce strong signals with suitable matrices. 

Zaima, N., Goto-Inoue, N., Adachi, K., and Setou, M. 2011. Selective analysis of lipids by thin-layer chromatography blot matrix-assisted laser desorption/ionization imaging mass spectrometry, J. Oleo ScL, 60 93-98. 

The results for bacterial whole-cell analysis described here establish the utility of MALDI-FTMS for mass spectral analysis of whole-cell bacteria and (potentially) more complex single-celled organisms. The use of MALDI-measured accurate mass values combined with mass defect plots is rapid, accurate, and simpler in sample preparation then conventional liquid chromatographic methods for bacterial lipid analysis. Intact cell MALDI-FTMS bacterial lipid characterization complements the use of proteomics profiling by mass spectrometry because it relies on accurate mass measurements of chemical species that are not subject to posttranslational modification or proteolytic degradation. 

To establish unambiguously the length of a hydrocarbon chain or the position of double bonds, mass spectral analysis of lipids or their volatile derivatives is invaluable. The chemical properties of similar lipids (for example, two fatty acids of similar length unsaturated at different positions, or two isoprenoids with different numbers of isoprene units) are very much alike, and their positions of elution from the various chromatographic procedures often do not distinguish between them. When the effluent from a chromatography column is sampled by mass spectrometry, however, the components of a lipid mixture can be simultaneously separated and identified by their unique pattern of fragmentation (Fig. 10-24). 

In lipidomics, MS can be used either by direct infusion, that is, by the so-called shotgun MS, or in combination with chromatographic separation, typically LC and sometimes also with GC. Both approaches have their own advantages and limitations. Most targeted lipid analyses are performed with liquid chromatography coupled to mass spectrometry (LC-MS), while the use of gas chromatography-mass spectrometry (GC-MS) is utilized only for the analysis of fatty acids and some steroids. In addition, surface analysis by MS has been applied in lipid analysis of intact tissues. 

Due to phase variation, there are fluctuations in expression levels of certain enzymes in bacteria, therefore, not all colonies or cells make the same structure of lipid A species. A micro-extraction method for extraction of lipid A from a single colony has been developed (Zhou et al., 2009). This method uses microwave-assisted enzymatic digestion and sodium acetate hydrolysis, suitable to analyze lipid A from both cell samples and an individual colony. Because the clean up of SDS is very time-consuming, and the contaminated SDS would seriously interfere with the analysis by mass spectrometry, the proteinase K, instead of SDS, is used to disrupt the cells. Using this method, the entire process for lipid A preparation only takes about 2 h with a detection limit to 1 (xg. 

Keusgen, M., Curtis, J.M. and Ayer, S.W. (1996) The use of nicotinates and sulfo-quinovosyl monoacylglycerols in the analysis of monounsaturated n-3 fatty acids by mass spectrometry. Lipids, 31 (2), 231-8. 

Brooks, P.W., Cardoso, J.N., Didyk, B., Eglinton, G., Humbertson, M.J. and Maxwell, J.R., 1977. Analysis of lipid fractions from environmental and geological sources by computerised gas chromatography/mass spectrometry. In R. Campos and J. Goni (Editors), Advances in Organic Geochemistry, 1975. Enadimsa, Madrid, pp. 433—453. 

Great advances have been made in the direct analysis of lipid extracts (i.e., those requiring minimal preparative fractionation) by the use of dedicated analytical instrumentation such as TLC-FID and HPLC. Mass spectrometry enables the combination of aspects of lipid separation by molecular weight and structural detail from techniques of ionization-induced fragmentation. Combined with chromatographic procedures, this represents the most powerful but expensive tool available for lipid analysis. 

The ability of PI synthetase to use 5-deoxy-5-fluoro-myo-inositol (4) as a substrate was confirmed by use of a radiolabeled compounds as shown in Figure 7. PI synthetase incorporated the analog into lipid in a time-dependent manner. The incorporation was absolutely dependent on the presence of CDP-diglyceride and was inhibited by the presence of myo-inositol (1) in the incubation mixture, as expected for PI synthetase. Chromatography of the reaction mixture revealed that a single radiolabeled product was formed with a mobility similar to, but distinct from, that of PI. Subsequent analysis has shown that the product is converted to a water-soluble form on mild alkaline hydrolysis and yields 5-deoxy-5-fluoro-myo-inositol (4) on treatment with phospholipase D, in agreement with the formation of phosphatidyl-5-deoxy-5-fluoro-myo-inositol as the product (data not shown). Determination of the absolute structure of these phospholipids awaits large-scale enzymatic synthesis, isolation of the product, and studies by mass spectrometry and NMR spectroscopy. 

Hyphenated TLC techniques. TLC has been coupled with other instrumental techniques to aid in the detection, qualitative identification and, occasionally, quantitation of separated samples, and these include the coupling of TLC with high-pressure liquid chromatography (HPLC/TLC), with Fourier transform infra-red (TLC/FTIR), with mass spectrometry (TLC/ MS), with nuclear magnetic resonance (TLC/NMR) and with Raman spectroscopy (TLC/RS). These techniques have been extensively reviewed by Busch (1996) and by Somsen, Morden and Wilson (1995). The chemistry of oils and fats and their TLC separation has been so well established that they seldom necessitate the use of these coupling techniques for their identification, although these techniques have been used for phospholipid detection. Kushi and Handa (1985) have used TLC in combination with secondary ion mass spectrometry for the analysis of lipids. Fast atom bombardment (FAB) has been used to detect the molecular species of phosphatidylcholine on silica based on the molecular ion obtained by mass spectrometry (Busch et al, 1990). 

Kushi, Y. and Handa, S. (1985) Direct analysis of lipids on thin layer plates by matrix-assisted secondary ion mass spectrometry. Journal of Biochemistry, 98 (1), 265-8. 

Jungalwala, F. B., Evans, J. E. and McCluer, R. H. (1984) Compositional and molecular species analysis of phospholipids by high performance liquid chromatography coupled with chemical ionisation mass spectrometry. J. Lipid Res., 25, 738-49. 

Kallio, H. and Currie, G. (1993b) Analysis of natural fats and oils by ammonia negative ion tandem mass spectrometry - triacylglycerols and positional distribution of their acyl groups, in CRC Handbook of Chromatography, Analysis of Lipids (eds K. Mukherjee, N. Weber and J. Sherma), CRC Press, Boca Raton, FL, pp. 435-58. 

Laakso, P. and Voutilainen, P. (1996) Analysis of triacylglycerols by silver ion high-performance atmospheric pressure chemical ionisation mass spectrometry. Lipids, 31, 1311-22. 

MacMillan, D. K. and Murphy, R. C. (1995) Analysis of lipid hydroperoxides and long-chain conjugated keto acids by negative-ion electrospray mass-spectrometry. J. Am. Soc. Mass Spectrom., 6, 1190-1201. 

The separation of intact polar lipids by liquid chromatography (LC) and the subsequent detection by mass spectrometry (MS) has today become straightforward. LC-MS is no longer a sophisticated technique only in the hands of specialists. Today, it is a routinely used, although advanced, analytical technique. The fields of application are expanding and today the use of LC-MS with electrospray (ES) ionization grows at the expense of other ionization techniques, at least where analysis of intact polar lipids is concerned. 

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