Rapid characterization of the fatty acyl composition of complex lipids by collision-induced dissociation time-of-flight mass spectrometry

Abstract

Profiling of leaf extracts from mutants of Arabidopsis with defects in lipid desaturation demonstrates the utility of collision-induced dissociation time-of-flight mass spectrometry (CID-TOF MS) for screening biological samples for fatty acid compositional alterations. CID-TOF MS uses the collision cell of a quadrupole time-of-flight mass spectrometer to simultaneously fragment all of the ions produced by an ionization source. Electrospray ionization CID-TOF MS in the negative mode can be used to analyze fatty acyl anions derived from complex lipids as well as free fatty acids. Although acyl anion yield is shown to be a function of the lipid class and the position on the glycerol backbone, acyl compositional profiles can be determined, and the TOF detector provides resolution of nominally isobaric acyl species in the profiles. Good precision is obtained when data are acquired for z1 min per sample.-Esch, S. W., P. Tamura Fatty acyl composition is most commonly analyzed by gas chromatography after derivatization to fatty acid methyl esters. When flame ionization or a mass spectrometer is used as the detector, quantitative compositional data are obtained. Liquid chromatography provides an alternative means of separation, in which fatty acids can be detected by ultraviolet light absorption after chemical conversion to an appropriate derivative form (1). Separation and analytical techniques include adsorption chromatography, silver nitrate chromatography, TLC, and HPLC (2). Most gas chromatography and liquid chromatography protocols for fatty acyl species are optimized for normal chain fatty acyl species. Other fatty acids, such as those containing heteroatoms (e.g., oxygen), often require specialized procedures for analysis. Plant oxylipins, for example, have been analyzed by GC-MS or liquid chromatography after various extraction and derivatization steps designed to enable detection and minimize the degradation of these often lowlevel compounds (3-7).Recently, we used a novel negative ion mode mass spectrometric scanning method for rapid profiling of free and ester-linked fatty acyl moieties in crude lipid extracts (8). The collision-induced dissociation time-of-flight (CID-TOF) experiment involved simultaneous collision-induced dissociation of all members of a complex mixture of lipids in the collision cell of a quadrupole time-of-flight (Q-TOF) instrument. The TOF analyzer provided accurate mass characterization of all intact acyl fragments in a single spectrum. Using this method as a "discovery scan," we identified fatty acyl groups, including several oxygenated species that increased in response to wounding of the plant Arabidopsis thaliana (8). Here, we describe this methodology and investigate the accuracy and precision of collision-induced dissociation time-of-flight mass spectrometry (CID-TOF MS).

Tocopherols Modulate Extraplastidic Polyunsaturated Fatty Acid Metabolism inArabidopsisat Low Temperature

“…Furthermore, the Q-TOF analysis indicated that these oxidized species, 18:4-O, 18:3-O, and 18:2-O, are the major and generally the only lipid fragments with nominal m/z of 291, 293, and 295 (Buseman et al, 2006;Esch et al, 2007). The level of oxylipin-containing lipid molecular species was quantified by precursor scanning of oxylipin acyl anions, m/z 291, 293, and 295, using a triple quadrupole mass spectrometer (API 4000; Applied Biosystems) in the negative mode.…”

New Applications of Mass Spectrometry in Lipid Analysis

“…To achieve this quantitative measure for this large number of molecular species, it was necessary to separate and to employ a different separation protocol that was ideal for each individual class of lipid. Lipidomic analyses of yeast (23), Caenorhabditis elegans (24), tuberculosis mycobacteria (25), human plasma (26), and others (27) have emerged.…”

Mutations in the Prokaryotic Pathway Rescue the fatty acid biosynthesis1 Mutant in the Cold

“…Lipids were rendered visible under UV light by exposure to 0.005% (w/v) primulin in 80% (v/v) acetone, after which the individual lipid classes were collected and their fatty acid content determined by gas chromatography. For lipidomics analysis, lipids were extracted according to the instructions provided by the Kansas Lipidomics Research Center (http://www.k-state.edu/lipid/lipidomics/), and PG molecular species were quantified by analysis performed at that facility (Esch et al, 2007). Lipid molecular species for the single lipids were reanalyzed, and the content of all molecular species in specific lipids was set as 100%.…”