Mass spectrometry advances in lipidomica: collision-induced decomposition of Kdo2-lipid A

Abstract

Mass spectrometry has made significant advances in the analysis of lipid substances, both simple and complex present in extracts of eukaryotic and prokaryotic cells. The development of the ionization techniques of electrospray ionization and matrix-assisted laser desorption ionization (MALDI) have both been applied to the analysis of lipids. The example of the types of structural information that can be obtained from MALDI-TOF tandem mass spectrometry is exemplified by the analysis of Kdo2-lipid A, a complex lipopolysaccharide known to activate toll-like 4 receptors on mammalian cells. Analysis of Kdo2-lipid A obtained from an Escherichia coli WBB06 was found to generate an abundant [M-H]- ion at m/z 2236.4 and a more abundant carbon-13 isotope at m/z 2237.4. Furthermore, collisional activation of the lipid A portion of the molecule at m/z 1796.3 resulted in a series of ions corresponding to the loss of all four fatty acyl groups as neutral carboxylic acids. An altogether different challenge of mass spectrometry applied to the area of lipid analysis is that of quantitative analysis. Two rather different requirements have emerged. One with high precision and accuracy for the measurement of relatively few lipid species that are produced at very low concentrations and typically interact with specific receptor proteins. A rather different challenge is that for the analysis of abundant lipid classes, which are composed of multiple molecular species that can approach several hundred under certain circumstances.

Figure 1

Matrix assisted laser desorption time-of-flight mass spectrometry of Kdo₂-lipid A (negative ions) using 6-aza-2-thiothymine in 10% ammonium citrate as matrix. Mass spectra were obtained in a tandem quadrupole time-of-flight mass spectrometer. Inset figures correspond to an expansion of the mass scales from m/z 2230 – 2550 and m/z 2200 – 2280, respectively.

Figure 2

Collision induced decomposition of the lipid A fragment ion m/z 1796.3 in a tandem quadrupole time-of-flight mass spectrometer using matrix assisted laser desorption ionization. The product ions correspond to the lipid A portion of the molecule (LA) and the corresponding loss of the fatty acids as free carboxylic acids as indicated in Scheme 2 as the simple fatty acids A, C, and D, and the complex fatty acid G. Other significant ions correspond to the loss of phosphoric acid moieties from the lipid A ion (LA) and individual fatty acyl groups.

Scheme 1

Kdo₂-Lipid A

Scheme 2