Quantification of Interactions between Serum Albumin and Endogenous Free Fatty Acids or Exogenous Chemicals by Stable Isotope-Coded Mass Spectrometry

Abstract

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 ...