title: Quantifying protein-fatty acid interactions using electrospray ionization mass spectrometry.
abstract: The application of the direct electrospray ionization mass spectrometry (ESI-MS) assay to quantify interactions between bovine β-lactoglobulin (Lg) and a series of fatty acids (FA), CH(3)(CH(2))(x)COOH, where x=6 (caprylic acid, CpA), 8 (capric acid, CA), 10 (lauric acid, LA), 12 (myristic acid, MA), 14 (palmitic acid, PA) and 16 (stearic acid, SA), is described. Control ESI-MS binding measurements performed on the Lg-PA interaction revealed that both the protonated and deprotonated gas phase ions of the (Lg + PA) complex are prone to dissociate in the ion source, which leads to artificially small association constants (K (a)). The addition of imidazole, a stabilizing solution additive, at high concentration (10 mM) increased the relative abundance of (Lg + PA) complex measured by ESI-MS in both positive and negative ion modes. The K(a) value measured in negative ion mode and using sampling conditions that minimize in-source dissociation is in good agreement with a value determined using a competitive fluorescence assay. The K (a) values measured by ESI-MS for the Lg interactions with MA and SA are also consistent with values expected based on the fluorescence measurements. However, the K (a) values measured using optimal sampling conditions in positive ion mode are significantly lower than those measured in negative ion mode for all of the FAs investigated. It is concluded that the protonated gaseous ions of the (Lg + FA) complexes are kinetically less stable than the deprotonated ions. In-source dissociation was significant for the complexes of Lg with the shorter FAs (CpA, CA, and LA) in both modes and, in the case of CpA, no binding could be detected by ESI-MS. The affinities of Lg for CpA, CA, and LA determined using the reference ligand ESI-MS assay, a method for quantifying labile protein-ligand complexes that are prone to in-source dissociation, were found to be in good agreement with reported values.
authors.affiliation: Alberta Ingenuity Centre for Carbohydrate Science and Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2G2.
authors.forename: Lan
authors.initials: L
authors.lastname: Liu
authors.forename: Elena N
authors.initials: EN
authors.lastname: Kitova
authors.forename: John S
authors.initials: JS
authors.lastname: Klassen
mesh_terms: D000085:Acetates; D000465:Algorithms; D000818:Animals; D002417:Cattle; D005227:Fatty Acids / Q000737:chemistry* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007782:Lactoglobulins / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
chemical_list: D000085:Acetates; D005227:Fatty Acids; D007782:Lactoglobulins; C018824:ammonium acetate