Identification and Quantitation of Fatty Acid Double Bond Positional Isomers: A Shotgun Lipidomics Approach Using Charge-Switch Derivatization

Abstract

The specific locations of double bonds in mammalian lipids have profound effects on biological membrane structure, dynamics and lipid second messenger production. Herein, we describe a shotgun lipidomics approach that exploits charge-switch derivatization with N-(4-aminomethylphenyl) pyridinium (AMPP) and tandem mass spectrometry for identification and quantification of fatty acid double bond positional isomers. Through charge-switch derivatization of fatty acids followed by positive-ion mode ionization and fragmentation analysis, a marked increase in analytic sensitivity (low fmol/μL) and the identification of double bond positional isomers can be obtained. Specifically, the locations of proximal double bonds in AMPP-derivatized fatty acids are identified by diagnostic fragment ions resulting from the markedly reduced 1,4-hydrogen elimination from the proximal olefinic carbons. Additional fragmentation patterns resulting from allylic cleavages further substantiated the double bond position assignments. Moreover, quantification of fatty acid double bond positional isomers is achieved by the linear relationship of the normalized intensities of characteristic fragment ions vs. the isomeric compositions of discrete fatty acid positional isomers. The application of this approach for the analysis of fatty acids in human serum demonstrated the existence of two double bond isomers of linolenic acid (i.e., Δ6,9,12 18:3, γ-linolenic acid (GLA), and Δ9,12,15 18:3, α-linolenic acid (ALA)). Remarkably, the isomeric ratio of GLA vs. ALA esterified in triglycerides was three-fold higher than the ratio of their non-esterified moieties. Through this developed method, previously underestimated or unidentified alterations in fatty acid structural isomers can be determined facilitating the identification of novel biomarkers and maladaptive alterations in lipid metabolism during disease.

Mass spectrometry-directed structure elucidation and total synthesis of ultra-long chain (O-acyl)-ω-hydroxy fatty acids

“…This approach has been reported to afford up to 20-fold increases in mass spectral and subjecting it to further CID (i.e., MS 3 ) gave rise to the spectrum shown in Figure 2A. This spectrum exhibited a near complete profile of carbon-carbon bond fission along the hydrocarbon chain that was consistent with prior reports of charge-remote fragmentation in AMPP-derivatized fatty acids (29).…”

“…N-(4-aminomethylphenyl)pyridinium (AMPP) was used to derivatize synthetic and meibum OAHFA using a commercially available kit, as previously described (28,29). 1-(3-(aminomethyl)-4-iodophenyl)pyridin-1-ium (4-I-AMPP) was used to prepare 4-I-AMPP-derivatised OAHFA by the same procedure.…”

Characterization of phthiocerol and phthiodiolone dimycocerosate esters of M. tuberculosis by multiple-stage linear ion-trap MS

“…By contrast, the traditional GC/MS method for identifi cation of mycocerosic acids is laborious, requiring acid methanolysis to form the methyl ester, separated by TLC, followed by reductive degradation to mycocerosic alcohol and another two-dimensional TLC purifi cation, before formation of the fi nal t-butyldimethylsilyl ether derivatives (23)(24)(25). The superb sensitivity gained for the acid detected as the AMPP derivative (as compared with its underivatized form) also facilitates structural identifi cation and quantitation ( 34,(40)(41)(42)(43). This aspect may deserve further investigation.…”

Rapid Identification and Quantification of Linear Olefin Isomers by Online Ozonolysis-Single Photon Ionization Time-of-Flight Mass Spectrometry

“…Recently, the technical advances in mass spectrometry (MS) [8], especially new ionization methods such as matrix-assisted laser desorption/ ionization (MALDI) [9] and electrospray ionization (ESI) [10], and multistage MS [11][12][13] have become powerful tools in overcoming the above mentioned difficulties. In addition, a variety of chemical derivatizations in conjunction with MS have also become effective methods for analysis of compounds with different double bond positions from following processes, such as dimethyl sulfide addition reaction (DSAR) [14], crossmetathesis (CM) [15,16], charge-switch derivatization (CSD) [17], and covalent adduct chemical ionization (CACI) [18,19].…”

“…In the method of CM, dichloromethane/methyl acrylate were first mixed to dissolute the target sample, and the secondgeneration Hoveyda-Grubbs catalyst was then added, followed by a stirring process, which lasted for 2-3 h [16]. Gross et al exploited a faster method by charge-switch derivatization with N-(4-aminomethylphenyl) pyridinium for 30 min, and then the derived fatty acid samples were injected into a tandem MS for analysis [17]. CACI, an online gas-phase derivatization, was developed to locate double bonds using acetonitrile as the reagent gas.…”