Mechanisms for ion formation during the electron impact-mass spectrometry of picolinyl ester and 4,4-dimethyloxazoline derivatives of fatty acids

Mass spectral studies have been conducted with isotopically stable labelled and fluorinated picolinyl esters and 4,4-dimethyloxazoline (DMOX) derivatives of fatty acids in order to establish mechanisms of ion formation. Reciprocal hydrogen transfer is shown to be involved in the formation of the ion at m/z 126 with dimethyloxazoline derivatives and for the ion at m/z 164 with picolinyl esters. Inclusion of a fluorine atom alpha to the carboxyl of a fatty acid has been demonstrated to enhance rearrangements for expulsion of internal chain fragments with both methyl ester and dimethyloxazoline derivatives. When two fluorine atoms are inserted into the alpha position a similar rearrangement has been shown to occur with picolinyl esters, although not nearly to the same extent as that observed with either of the other derivatives. Mechanisms for such rearrangements are proposed and discussed. With fatty acid dimethyloxazoline derivatives the M-15 ion arises solely from the loss of a methyl radical from the ring and the M-43 ion has at least three different mechanisms of formation. Such rearrangements make it difficult to establish the identity of the terminal moiety of the alkyl chain. In mass spectrometry terms the picolinyl ester would seem to be the superior derivative for structural characterisation of fatty acids.     

Practical aspects of liquid chromatography-mass spectrometry (LS-MS) of lipids

Techniques for coupling liquid chromatography with mass spectrometry are reviewed and an interface is described for the analysis of lipids by mass spectrometry. The interface for coupling liquid chromatography with mass spectrometry for lipid analysis is based on the moving wire transport principle using an endless stainless steel belt of novel construction. After evaporation of the solvent, the solute remains as a residue on the belt which transports it into a reactor where it is volatilized by evaporation or conversion to hydrocarbons. The volatile compounds are then fed into the source of a chemical ionization mass spectrometer for mass analysis by total or single ion monitoring as well as for structural identification or compositional analysis. The sensitivity of the system was approx. 1 mg per component separated in the eluate of a high efficiency column. The capabilities of the interface were demonstrated by its application to reference compounds representative of triglycerides, sterols, steryl esters, glyceryl ethers, glyceryl ether diesters, glycerophosphatides, sphingolipids, prostaglandins and fatty acid methyl esters. It also was applied to the analysis of methyl ester ozonides to demonstrate the use of LC-MS for the localization of double bonds in unsaturated fatty acids.     

Synthesis of saturated long chain fatty acids from sodium acetate-1-C14 by Mycoplasma

Three strains of Mycoplasma, M. laidlawii A and B, and Mycoplasma sp. A60549, were grown in broth containing sodium acetate-1-C(14). The methyl esters of the phospholipid fatty acids of harvested radioactive cells were prepared and identified by comparison of their mobilities to known radioactive fatty acid methyl esters by use of a modified reversed-phase partition-thin layer chromatographic technique. No radioactive methyl oleate or methyl linoleate was detected. Compounds migrating as radioactive methyl myristate, stearate, palmitate, and, with less certainty, laurate and octanoate were detected. The qualitative findings for all three organisms appeared similar. M. laidlawii B synthesized a radioactive substance, presumably a saturated fatty acid detected as the methyl ester derivative, which migrated in a position intermediate to methyl myristate-1-C(14) and methyl palmitate-1-C(14). This work indicates that M. laidlawii A and B and Mycoplasma sp. A60549 are capable, in a complex medium containing fatty acids, of synthesizing saturated but not unsaturated fatty acids entirely or in part from acetate.     

The Collection and Elution of Radioactive Fatty Acid Methyl Esters During Quantitative Gas-Liquid Chromatography

Abstract

An improved procedure is described for the collection and elution of low levels of radioactive fatty acid methyl esters separated by gas-liquid chromatography. A gas chromatographic effluent splitter was employed to partition fatty acid methyl ester samples in the column effluent, Condensation of a portion of the eluted fatty acids was accomplished in borosilicate glass tubing collectors maintained at ?70°C, Quantitation of nanomolar levels of fatty acid methyl esters was accomplished by calibrating the gas chromatographic flame ionization detectors with the splitters opened or closed. The elution of condensed radioactive fatty acid methyl esters from the glass collectors was complete when benzene followed by a toluene based scintillation fluid were employed as solvents. The method described may be applicable to the analysis of cis-trans isomers of unsaturated fatty acids.     

Biocatalytic synthesis of ascorbyl esters and their biotechnological applications

Ascorbyl fatty acid esters act both as antioxidants and surfactants. These esters are obtained by acylation of vitamin C using different acyl donors in presence of chemical catalysts or lipases. Lipases have been used for this reaction as they show high regioselectivity and can be used under mild reaction conditions. Insolubility of hydrophilic ascorbic acid in non-polar solvents is the major obstacle during ascorbic acid esters synthesis. Different strategies have been invoked to address this problem viz. use of polar organic solvents, ionic liquids, and solid-phase condensation. Furthermore, to improve the yield of ascorbyl fatty acid esters, reactions were performed by (1) controlling water content in the reaction medium, (2) using vacuum to remove formed volatile side product, and (3) employing activated acyl donors (methyl, ethyl or vinyl esters of fatty acids). This mini-review offers a brief overview on lipase-catalyzed syntheses of vitamin C esters and their biotechnological applications. Also, wherever possible, technical viability, scope, and limitations of different methods are discussed.     

Location of double bonds and cyclopropane rings in fatty acids by mass spectrometry

Electron-impact mass spectrometric procedures for locating the position of double bonds and cyclopropane rings in long-chain fatty acids are reviewed. Since unsaturation is not located directly by mass spectrometry, the properties of suitable derivatives are summarized. Epoxides are readily prepared from double bonds and on opening of the ring with various reagents useful derivatives are obtained, the most promising to date being hydroxymethoxy esters whose trimethylsilyl ethers give good mass spectra. Trimethylsilyl ethers of vicinal diols, prepared by direct hydroxylation, are recommended for the analysis of polyunsaturated fatty acid esters using combined gas chromatography-mass spectrometry. Oxymercuration-demercuration techniques are very convenient and one particular procedure can specifically locate unsaturation up to five carbons distant from the carboxyl group. An alternative approach enables the location of double bonds and cyclopropane rings in fatty acids by direct mass spectrometry of pyrrolidides. Cyclopropane rings can be positively located in fatty acid esters by mass spectrometry of isomeric ketones or methoxy derivatives prepared by chromium trioxide oxidation on poron trifluoride-catalysed methoxylation, respectively. A variety of other procedures are also considered and some guidelines are given for choosing a method to suit a particular unsaturated acid.     

The N-terminal SH4 region of the Src family kinase Fyn is modified by methylation and heterogeneous fatty acylation: role in membrane targeting, cell adhesion, and spreading

The N-terminal SH4 domain of Src family kinases is responsible for promoting membrane binding and plasma membrane targeting. Most Src family kinases contain an N-terminal Met-Gly-Cys consensus sequence that undergoes dual acylation with myristate and palmitate after removal of methionine. Previous studies of Src family kinase fatty acylation have relied on radiolabeling of cells with radioactive fatty acids. Although this method is useful for verifying that a given fatty acid is attached to a protein, it does not reveal whether other fatty acids or other modifying groups are attached to the protein. Here we use matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry to identify fatty acylated species of the Src family kinase Fyn. Our results reveal that Fyn is efficiently myristoylated and that some of the myristoylated proteins are also heterogeneously S-acylated with palmitate, palmitoleate, stearate, or oleate. Furthermore, we show for the first time that Fyn is trimethylated at lysine residues 7 and/or 9 within its N-terminal region. Both myristoylation and palmitoylation were required for methylation of Fyn. However, a general methylation inhibitor had no inhibitory effect on myristoylation and palmitoylation of Fyn, suggesting that methylation occurs after myristoylation and palmitoylation. Lysine mutants of Fyn that could not be methylated failed to promote cell adhesion and spreading, suggesting that methylation is important for Fyn function.     

Gas-liquid chromatography of the heptafluorobutyrate derivatives of the O-methyl-glycosides on capillary columns: a method for the quantitative determination of the monosaccharide composition of glycoproteins and glycolipids

We have developed a method involving the formation of hepta-fluorobutyrate derivatives of O-methyl-glycosides liberated from glycoproteins and glycolipids following methanolysis. The stable derivatives of the most common monosaccharides of these glycoconjugates (Ara, Rha, Xyl, Fuc, Gal, Man, Glc, GlcNAc, GalNAc, Neu5Ac, KDN) can be separated and quantitatively and reproducibly determined with a high degree of sensitivity level (down to 25 pmol) in the presence of lysine as an internal standard. The GlcNAc residue bound to Asn in N-glycans is quantitatively recovered as two peaks. The latter were easily distinguished from the other GlcNAc residues of N-glycans, thus allowing a considerable improvement of the data on structure of N-glycans obtained from a single carbohydrate analysis. The most common contaminants present in buffers commonly used for the isolation of soluble or membrane-bound glycoproteins (SDS, Triton X-100, DOC, TRIS, glycine, and polyacrylamide or salts, as well as monosaccharide constituents of proteoglycans or degradation products of nucleic acids) do not interfere with these determinations. A carbohydrate analysis of glycoproteins isolated from a SDS/PAGE gel or from PDVF membranes can be performed on microgram amounts without significant interferences. Since fatty acid methyl esters and sphingosine derivatives are separated from the monosaccharide peaks, the complete composition of gangliosides can be achieved in a single step starting from less than 1 microg of the initial compound purified by preparative Silicagel TLC. Using electron impact ionization mass spectrometry, reporter ions for the different classes of O-methyl-glycosides (pentoses, deoxy-hexoses, hexoses, hexosamines, uronic acids, sialic acid, and KDN) allow the identification of these compounds in very complex mixtures. The mass of each compound can be determined in the chemical ionization mode and detection of positive or negative ions. This method presents a considerable improvement compared to those using TMS derivatives. Indeed the heptafluorobutyrate derivatives are stable, and acylation of amino groups is complete. Moreover, there is no interference with contaminants and the separation between fatty acid methyl-esters and O-methyl glycosides is achieved.     

Fatty acids of astaxanthin esters in krill determined by mild mass spectrometry

Krill is a major source of astaxanthin, which has strong antioxidant activity. Fractions with astaxanthin monoesters and diesters of Antarctic krill Euphausia superba were isolated. Astaxanthin esters were separated by C18-HPLC depending on the number of carbons and double bonds of esterified fatty acid(s). Small amounts of other lipids remained in the samples, but relative molecular masses of carotenoid esters could be measured by field desorption mass spectrometry without fragmentation and interference from contaminant lipids. The fatty acids were determined by calculation of difference between astaxanthin and astaxanthin esters. Only five kinds of fatty acids, dodecanoate, tetradecanoate, hexadecanoate, hexadecenoate and octadecenoate, were detected. Fast atom bombardment mass spectrometry and secondary ion mass spectrometry showed similar spectra. The fatty acid composition in astaxanthin esters was different from those in krill lipids. Therefore, determination of fatty acids in carotenoid esters by a combination of HPLC elution profile and mild mass spectrometry is found to be a useful tool.     

Analysis of fatty acids from lipopolysaccharides of Bacteroides thetaiotaomicron and Bacteroides fragilis]

The aim of this study was to determine and to compare fatty acids occurring in lipopolysaccharides (LPS) isolated from B. thetaiotaomicron and B. fragilis strains of different origin. Lipopolysaccharides of three B. thetaiotaomicron strains and four B. fragilis strains were isolated by phenol-water extraction according to the procedure of Westphal and Jann (1965). Water-phase LPS fractions were then treated with nucleases and purified by ultracentrifugation as described by Gmeiner (1975). Fatty acid methyl esters, obtained by methanolysis of LPS, were analysed in gas-liquid chromatography combined with mass spectrometry (GLC-MS). Trimethylsilylated hydroxyl groups of fatty acid methyl esters were identified with GLC-MS using a method of selective ion monitoring (SIM). Lipopolysaccharides of B. thetaiotaomicron and B. fragilis strains contained long-chain (15-18 carbon atoms) fatty acids. The broad spectrum of simple long-chain and branched-chain fatty acids as well as 3-hydroxy fatty acids were detected. The main fatty acid of analyzed bacterial species was 3-hydroxy-hexadecanoic acid (3OH C16:0). Several 3-hydroxy fatty acids were detected in LPS of examined strains. Fatty acids occurring in LPS of B. thetaiotaomicron and B. fragilis strains appeared to be qualitatively similar. Quantitative differences in fatty acids composition of lipopolysaccharides isolated from strains of different origin were observed.     

Heterogeneous fatty acylation of Src family kinases with polyunsaturated fatty acids regulates raft localization and signal transduction.

Fatty acylation of Src family kinases is essential for localization of the modified proteins to the plasma membrane and to plasma membrane rafts. It has been suggested that the presence of saturated fatty acyl chains on proteins is conducive for their insertion into liquid ordered lipid domains present in rafts. The ability of unsaturated dietary fatty acids to be attached to Src family kinases has not been investigated. Here we demonstrate that heterogeneous fatty acylation of Src family kinases occurs and that the nature of the attached fatty acid influences raft-mediated signal transduction. By using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, we show that in addition to 14:0 (myristate), 14:1 and 14:2 fatty acids can be attached to the N-terminal glycine of the Src family kinase Fyn when the growth media are supplemented with these dietary fatty acids. Moreover, we synthesized novel iodinated analogs of oleate and stearate, and we showed that heterogeneous S-acylation can occur on cysteine residues within Fyn as well as Galpha, GAP43, and Ras. Modification of Fyn with unsaturated or polyunsaturated fatty acids reduced its raft localization and resulted in decreased T cell signal transduction. These studies establish that heterogeneous fatty acylation is a widespread occurrence that serves to regulate signal transduction by membrane-bound proteins.     

Alpha and beta subunits of the nicotinic acetylcholine receptor contain covalently bound lipid

Labeling of the BC3H1 muscle-like cell line with [3H] palmitate, followed by immunoprecipitation of the acetylcholine receptor, indicated that the alpha and beta subunits of the receptor contain covalently bound fatty acid. After acid hydrolysis, fatty acid methyl esters could be recovered from the isolated [3H]palmitate-labeled alpha subunit. Treatment of differentiated BC3H1 cells with cerulenin, an inhibitor of fatty acid and sterol synthesis and fatty acid acylation of proteins, resulted in a 50% inhibition in expression of the acetylcholine receptor on the cell surface under conditions where there was minimal inhibition of protein synthesis. We conclude that this previously undetected post-translational modification may play a role in assembly and/or surface expression of the acetylcholine receptor.     

Improved detection of polyunsaturated fatty acids as phenacyl esters using liquid chromatography-ion trap mass spectrometry

The fatty acid composition of Shewanella pealeana was determined by the analysis of fatty acid methyl esters via gas chromatography-mass spectrometry (GC-MS) and fatty acid 2-oxo-phenylethyl esters via high-performance liquid chromatography-mass spectrometry (LC-MS) combined with ultra violet (UV) detection. There was good agreement between the percentage composition of components determined by GC-MS and LC-UV analyses. However, LC-MS analysis using Atmospheric Pressure Chemical Ionization (APCI) demonstrated dramatically enhanced detection of unsaturated fatty acid 2-oxo-phenylethyl esters. The degree of enhancement was proportional to the degree of unsaturation. Tests with a pure polyunsaturated fatty acid (PUFA) standard gave an absolute detection limit in full scan mode of 200 pg. In samples, the selectivity of MS over UV gave a significantly lower detection limit due to lack of chemical interferences. In 'Selected Reaction Monitoring' (SRM) mode, the detection limit was 5 pg. This was essentially independent of whether the sample is a standard or complex mixture of fatty acids. Tandem mass spectrometry was used to support structural information and to enhance the ability to target specific fatty acids. Several PUFAs which were not evident from GC-MS analysis were detected and identified by APCI LC-MS, including some rare or novel PUFAs from S. pealeana and a menhaden oil standard. Detailed analysis of bacterial fatty acid composition by either GC-MS or APCI LC-MS is highly preferable to analysis systems based solely on retention time identification.     

Evidence in the formation of conjugated linoleic acids from thermally induced 9t12t linoleic acid: a study by gas chromatography and infrared spectroscopy

Thermally induced isomerisation leading to the formation of conjugated linoleic acids (CLAs) has been observed for the first time during the thermal treatment of 9t12t fatty acid triacylglycerol, and methyl ester. Fifteen microlitre portions of the triacylglycerol sample containing 9t12t fatty acid (trilinoelaidin) were placed in micro glass ampoules and sealed under nitrogen, then subjected to thermal treatment at 250 °C. The glass ampoules were removed at regular time intervals, cut open, and the contents were analysed by infrared spectroscopy using a single reflectance attenuated total internal reflectance crystal accessory. The samples were then subjected to derivatisation into their methyl esters. The methyl esters of the isomerised fatty acids were analysed by gas chromatography. The same procedure was repeated with methyl ester samples containing 9t12t fatty acid (methyl linoelaidate). Each sample was subjected to infrared measurements and gas chromatographic analysis after appropriate dilution in heptane.The results show that the thermally induced isomerisation of 9t12t fatty acids from both triacylglycerol molecules and methyl esters give identical CLA profiles as those found for the thermally induced isomerisation of 9c12c fatty acids. The infrared spectrometry provides additional evidence confirming the formation of CLA acids during thermal treatment. A mechanism for the formation of the CLAs from 9t12t fatty acid molecules is also formulated for the first time. This mechanism complements the pathways of formation of CLAs from 9c12c fatty acids during thermal treatment.     

Estimation of plasma free fatty acids as the trimethylsilyl (TMS) esters

Quantitative estimates of free fatty acids in total lipid extracts of plasma were obtained by glc on nonpolar columns following trimethylsilylation. The presence of other lipid esters in the reaction mixture had no effect upon the yield of the trimethylsilyl (TMS) esters or upon their resolution on the glc column. Routine quantitations by gas-liquid chromatography (glc) were obtained on 2 ft × 1/8 in. o.d. stainless steel columns packed with 3% OV-1 on 100–120 mesh Gas Chrom Q by means of temperature programming in the range 175–350°C with tridecanoin as internal standard. High resolution glc of the TMS esters of fatty acids was done on a 6 ft × 1/8 in. o.d. glass column packed with 1% SE-30 on Gas Chrom Q. In both instances the fatty acids were resolved on the basis of carbon number and by the presence or absence of double bonds. On gas chromatography/mass spectrometry (GC/MS), TMS esters of fatty acids were shown to yield proportionally greater amounts of high mass fragments, including the parent ions, than their methyl or ethyl esters, which has special advantages for the detection and characterization of polyunsaturated fatty acids.     

Lipidomic approaches to the study of phospholipase A2-regulated phospholipid fatty acid incorporation and remodeling

The distribution of fatty acids among cellular glycerophospholipids is finely regulated by the CoA-dependent acylation of lysophospholipids followed by transacylation reactions. Arachidonic acid is the fatty acid precursor of a wide family of bioactive compounds called the eicosanoids, with key roles in innate immunity and inflammation. Because availability of free AA constitutes a rate-limiting step in the generation of eicosanoids by mammalian cells, many studies have been devoted to characterize the processes of arachidonate liberation from phospholipids by phospholipase A₂s and its re-incorporation and further remodeling back into phospholipids by acyltransferases and transacylases. These studies have traditionally been conducted by using radioactive precursors which do not allow the identification of the phospholipid molecular species involved in these processes. Nowadays, lipidomic approaches utilizing mass spectrometry provide a new frame for the analysis of unique phospholipid species involved in fatty acid release and phospholipid incorporation and remodeling. This review focuses on the mass spectrometry techniques applied to the study of phospholipid fatty acid trafficking and the recent advances that have been achieved by the use of this technique.     

Detection of lactobacillic acid in low erucic rapeseed oil—A note of caution when quantifying cyclic fatty acid monomers in vegetable oils

The purpose of this work was to identify an unknown component which has been detected during the analysis of cyclic fatty acid monomers (CFAMs) in low erucic acid rapeseed oils (LEAR). A sample of crude LEAR was transformed into fatty acid methyl esters (FAMEs) and hydrogenated using PtO₂. The hydrogenated sample was fractionated by reversed-phase high-performance liquid chromatography (RP-HPLC) and the fraction containing the CFAMs transformed into picolinyl esters. Analysing these picolinyl derivatives by gas–liquid chromatography coupled to mass spectrometry (GC–MS) showed that the unknown product observed in LEAR is the 11,12-methylene-octadecanoic acid. This cyclic fatty acid was also found in crude LEAR and in the corresponding seeds but was not detected in crude soya and sunflower oils. As this acid is present in the same fraction as CFAMs, known to be formed during heat treatment, great care must therefore be taken for not including it when quantifying CFAMs. It is thus necessary to verify by mass spectrometry the structures of the CFAMs in the isolated cyclic fatty acid fraction prior to quantification.     

Determination of double bond positions in fatty acids with conjugated double bonds

The positions of the double bonds in fatty acids with conjugated double bonds may be determined by mass spectrometry of the methyl esters of their trimethylsilyl ether derivatives obtained by hydroxylation of the double bonds followed by silylation of the resulting polyols. The method has been applied to trans-9,trans-11- and trans-10, trans-12-octadecadienoic acid.     

Studies on Moss Spores. IV. Mass Spectrometric Identification of Saturated and Monoenoic Long Chain Fatty Acids in the Triglyceride Fraction of Polytrichum commune Spores

The saturated long chain fatty acid methyl esters of the triglyceride fraction of Polytrichum commune spores were separated by silver nitrate TLC and identified by a combination of gas chromatographic-mass spectrometric technique. The saturated fatty acid methyl esters were straight-chained, and even-numbered with carbon numbers ranging from 12 to 26 or odd-numbered with carbon numbers ranging from 13 to 25. The major components of the fraction containing saturated fatty acid methyl esters were methyl palmitate and methyl stearate. The fatty acid methyl esters of the monoenoic fraction isolated by silver nitrate TLC were converted to TMSO derivates which were analysed by gas chromatography-mass spectrometry. The analysis gave evidence of positional isomers. The fraction contained the following straight chain monoenoic fatty acid methyl ester isomers: methyl 7-cis-hexadecenoate, methyl 9-cis-hexadecenoate, methyl 9-cis-heptadecenoate, methyl 9-cis-octadecenoate, methyl 11-cis-octadecenoate, and methyl 11-cis-eicosenoate. The major components were methyl 9-cis-octadecenoate and methyl 7-cis-hexadecenoate.     

Identification of conjugated linoleic acid elongation and beta-oxidation products by coupled silver-ion HPLC APPI-MS

Atmospheric pressure photoionisation (APPI) was used in combination with silver-ion (Ag(+))-HPLC for detection of (conjugated) fatty acid methyl esters (FAME) by tandem-mass spectrometry. APPI-MS of methyl esters of conjugated linoleic acid showed an increase in signal-to-noise ratio by a factor of 40 compared to atmospheric pressure chemical ionization in the positive mode. It was possible to identify double bond position, configuration and chain length of FAME based on chromatographic separation and mass detection. The developed LC-MS method is useful for the analysis of CLA elongation and beta-oxidation products, especially with trans,trans-configuration, which are difficult to analyze by conventional GC-MS techniques.