Abstract
Triacylglycerols (TAGs) isolated from a biological sample provide a challenge for mass spectrometric analysis because of the complexity of naturally occurring TAGs, which may contain different fatty acyl substituents resulting in a large number of molecular species having the identical elemental composition. We have investigated the use of mass spectrometry to obtain unambiguous information as to the individual TAG molecular species present in a complex mixture of triacylglycerols using a linear ion trap mass spectrometer. Ammonium adducts of TAGs, [M+NH4]+, were generated by electrospray ionization, which permitted the molecular weight of each TAG molecular species to be determined. The mechanisms involved in the decomposition of the [M+NH4]+ and subsequent fragment ions were investigated using deuterium labeling, MS/MS, and MS3 experiments. Collision induced decomposition of [M+NH4]+ ions resulted in the neutral loss of NH3 and an acyl side-chain (as a carboxylic acid) to generate a diacyl product ion. MS/MS data were used to identify each acyl group present for a given [M+NH4]+ ion, and this information could be combined with molecular weight data to identify possible TAG molecular species present in a biological extract. Subsequent MS3 experiments on the resultant diacyl product ions, which gave rise to acylium (RCO+) and related ions, enabled unambiguous TAG molecular assignments. These strategies of MS, MS/MS, and MS3 experiments were applied to identify components within a complex mixture of neutral lipids extracted from RAW 264. 7 cells.
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Coleman, R. A.; Lee, D. P. Enzymes of triacylglycerol synthesis and their regulation. Prog. Lipid Res 2004, 43, 134–176.
Salter, A. M.; Brindley, D. N. The biochemistry of lipoproteins. J. Inherit. Metab. Dis 1988, 1, 4–17.
Gibbons, G. F. Related Assembly and secretion of hepatic very low density lipoprotein. Biochem. J 1990, 268, 1–13.
Yeaman, S. J. Hormone-sensitive lipase—new roles for an old enzyme. Biochem. J 2004, 379, 11–22.
Weiss, R.; Dziura, J.; Burgert, T. S.; Tamborlane, W. V.; Taksali, S. E.; Yeckel, C. W.; Allen, K.; Lopes, M.; Savoye, M.; Morrison, J.; Sherwin, R. S.; Caprio, S. Obesity and the metabolic syndrome in children and adolescents. N. Engl. J. Med 2004, 350, 2362–2374.
Goodpaster, B. H.; Wolf, D. Skeletal muscle lipid accumulation in obesity, insulin resistance, and type 2 diabetes. Pediatr. Diabetes 2004, 5, 219–226.
Large, V.; Peroni, O.; Letexier, D.; Ray, H.; Beylot, M. Metabolism of lipids in human white adipocyte. Diabetes Metab 2004, 30, 294–309.
Liu, P.; Ying, Y.; Zhao, Y.; Mundy, D. I.; Zhu, M.; Anderson, R. G. Chinese hamster ovary K2 cell lipid droplets appear to be metabolic organelles involved in membrane traffic. J. Biol. Chem. 2004, 279, 3787–3792.
Murata, T. Analysis of triglycerides by gas chromatography/chemical ionization mass spectrometry. Anal. Chem. 1977, 49, 2209–2213.
Murata, T.; Takahashi, S. Analysis of triglyceride mixtures by gas chromatography-mass spectrometry. Anal. Chem. 1973, 45, 1816–1823.
Hites, R. A. Mass spectrometry of triglycerides. Methods Enzymol 1975, 35, 348–359.
Sjovall, O.; Kuksis, A.; Marai, L.; Myher, J. J. Elution factors of synthetic oxotriacylglycerols as an aid in identification of peroxidized natural triacylglycerols by reverse-phase high-performance liquid chromatography with electrospray mass spectrometry. Lipids 1997, 32, 1211–1218.
Duffin, K. L.; Henion, J. D.; Shieh, J. J. Electrospray and tandem mass spectrometric characterization of acylglycerol mixtures that are dissolved in nonpolar solvents. Anal. Chem. 1991, 63, 1781–1788.
Hvattum, E. Analysis of triacylglycerols with nonaqueous reversed-phase liquid chromatography and positive ion electrospray tandem mass spectrometry. Rapid Commun. Mass Spectrom. 2001, 15, 187–190.
Holcapek, M.; Jandera, P.; Zderadicka, P.; Hruba, L. Characterization of triacylglycerol and diacylglycerol composition of plant oils using high-performance liquid chromatography-atmospheric pressure chemical ionization mass spectrometry. J. Chromatogr. A. 2003, 1010, 195–215.
Han, X.; Gross, R. W. Quantitative analysis and molecular species fingerprinting of triacylglyceride molecular species directly from lipid extracts of biological samples by electrospray ionization tandem mass spectrometry. Anal. Biochem. 2001, 295, 88–100.
Hsu, F. F.; Turk, J. Structural characterization of triacylglycerols as lithiated adducts by electrospray ionization mass spectrometry using low energy collisionally activated dissociation on a triple stage quadrupole instrument. J. Am. Soc. Mass Spectrom. 1999, 10, 587–599.
Byrdwell, W. C.; Neff, W. E. Dual parallel electrospray ionization and atmospheric pressure chemical ionization mass spectrometry (MS), MS/MS, and MS/MS/MS for the analysis of triacylglycerols and triacylglycerol oxidation products. Rapid Commun. Mass Spectrom. 2002, 16, 300–319.
Cheng, C.; Gross, M. L.; Pittenauer, E. Complete structural elucidation of triacylglycerols by tandem sector mass spectrometry. Anal. Chem. 1998, 70, 4417–4426.
Bligh, E. G.; Dyer, W. J. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 1959, 37, 911–917.
Kaluzny, M. A.; Duncan, L. A.; Merritt, M. V.; Epps, D. E. Rapid separation of lipid classes in high-yield and purity using bonded phase columns. J. Lipid Res. 1985, 26, 135–140.
Hamilton, J. G.; Comai, K. Separation of neutral lipids and free fatty acids by high-performance liquid chromatography using low wavelength ultraviolet detection. J. Lipid Res. 1984, 25, 1142–1148.
Stuebiger, G.; Pittenauer, E.; Allmaier, G. Characterization of castor oil by on-line and off-line nonaqueous reverse-phase high-performance liquid chromatography-mass spectrometry (APCI and UV/MALDI). Phytochem. Anal. 2003, 14, 337–346.
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Published online July 12, 2005
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McAnoy, A.M., Wu, C.C. & Murphy, R.C. Direct qualitative analysis of triacylglycerols by electrospray mass spectrometry using a linear ion trap. J Am Soc Mass Spectrom 16, 1498–1509 (2005). https://doi.org/10.1016/j.jasms.2005.04.017
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DOI: https://doi.org/10.1016/j.jasms.2005.04.017