Fatty acids covalently bonded with other molecules have been implicated in many important biological processes. We describe here a rapid approach termed isotope-coded fatty acid transmethylation (iFAT) that integrates extraction, transmethylation, and isotopic labeling into a single step with the aid of ultrasonic irradiation for comparative analysis of fatty acids by mass spectrometry. In this approach, samples without any prefractionation were mixed with a methanol solution of 0.5 M NaOH and an n-hexane solution. The intense wave shocks and cavitations generated by ultrasonic irradiation not only speed the alkaline-catalyzed transmethylation reaction but also facilitate the simultaneous mass transfer of fatty acid methyl esters into the top n-hexane extraction phase that was injected into a GC/MS system. By using commercially available d(3)-methanol, we were able to compare the intensity of labeled and unlabeled methyl esters and their corresponding fragment ions. The detection limit can be down to the picogram level. Major advantages of the iFAT strategy are summarized in the following: (1) Efficient heterogeneous reactions. Solid samples such as dried cell lysates or detergent-resistant fractions can be readily transformed and analyzed with the aid of ultrasound irradiation. (2) Accurate quantification of fatty acids. Evaluation of the completeness or losses of transformation reactions across lipid classes has been hampered due to a lack of suitable methods. Isotope labeling can be used as an internal standard for accurate comparison of the fatty acid composition in different cell states. (3) Reduced interferences from complex biological context. The iFAT strategy not only differentially labels fatty acids in different samples, but also volatilizes those molecules, and thus, they are isolated from the bulk background and analyzed by GC/MS. This proposed approach has been applied to quantitatively determine the fatty acid composition in plant oil and in budding yeast cell lysates and detergent-resistant fractions. It should provide a widely applicable means for quantitative comparison of the fatty acid composition in cells and tissues.
S erum albumin is the most abundant blood protein synthesized in the liver. In addition to the function of maintaining colloid osmotic pressure, serum albumin plays important roles in transporting, distributing, and metabolizing many endogenous or exogenous chemicals.1 Binding of serum albumin with those compounds determines their plasma concentration and thus subsequent physiological or toxic effects.2,3 Crystallographic studies 4À7 have shown the structure of this protein in the solid state. However, the three-dimensional structure of serum albumin in aqueous solution and its contribution to binding with various ligands remain largely unknown. Although NMR (nuclear magnetic resonance) 8À10 and fluorescence spectroscopy 11 can provide direct experimental data in solution, the high molecular weight of albumin protein as well as the complication of data interpretation limit their global applications. Recently, with the availability of the genome database, computational modeling based on primary amino acid sequences has emerged as a useful tool to evaluate the binding possibility.12,13 But every kind of reversible or permanent protein posttranslational modification, such as attachment of small organic groups, cleavage of signal peptides, formation of disulfide bridges, as well as complex unknown physiological status, cannot be predicted from the genome, and it is thus difficult to incorporate these into the computational modeling. The situation is even more difficult for proteins isolated from species that do not have genome databases. Therefore, it is necessary to develop efficient solution-based techniques that can quantitatively describe how the structure of serum albumin determines the interactions with different ligands.Mass spectrometry-based methods 14 have attracted considerable attention in a broad range of research areas because of their high sensitivity, resolution, and throughput. In this work, the interactions between serum albumin and endogenous free fatty acids as well as exogenous pesticide residues have been investigated by a mass spectrometric approach based on stable isotopecoded free fatty acid methylation (iFFAM). Because free fatty acids are primary endogenous ligands of serum albumin, 15 quantification of variations of unbound free fatty acids in solution under different conditions directly indicates the binding status of serum albumin with exogenous ligands.The iFFAM approach was based on the equilibrium partition between n-hexane and aqueous phases. The principle and major procedures are summarized as follows. In this work, the aqueous solutions containing either native/denatured albumin or the mixture of albumin and exogenous chemicals were incubated with n-hexane solutions. Free fatty acids in n-hexane phases of the control and the sample were derived with a d0-or d3-methanol solution of 4% HCl, respectively. The resultant d0-and d3-fatty acid methyl esters were equally mixed together and analyzed by GC-MS. The original ratios of free fatty acids in the sample over that in the control ...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.