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 insource 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.

Reliable Determinations of Protein–Ligand Interactions by Direct ESI-MS Measurements. Are We There Yet?

“…Furthermore, different instruments sometimes produce divergent results for the same interaction. However, over the past decade, a number of methodological advances have significantly improved the reliability and applicability of the ESI-MS assay, and affinities measured for a multitude protein-ligand complexes, as well as other types of biological complexes have been shown to be in agreement with values determined by other analytical methods [14][15][16][17][18][19][20][21][22][23]. So, does this mean that the ESI-MS assay has matured to the point where the technique can be reliably used by researchers who do not have extensive training in MS?…”

“…However, it is important to note that the gas-phase stabilities of PL complexes generally do not parallel the solution binding affinities. For example, some PL complexes, which are stabilized by strong ionic interactions in solution, exhibit low gas phase stabilities [47], while some PL complexes formed by hydrophobic bonding are quite stable in the gas phase [23,50,51]. Collisional heating of gaseous ions may occur at various stages during the ion sampling process, such as within the heated metal sampling capillary (if used), in the nozzle (or orifice)-skimmer region, and during accumulation of ions within external rf multipole storage devices (e.g., hexapole) [14,36,47,50,51].…”

“…In cases where gentle sampling conditions do not eliminate the occurrence of in-source dissociation, the employment of stabilizing additives may prove beneficial. For example, the addition of imidazole to solution, at high concentration (91 mM), has been shown to prevent gas phase dissociation of the ions of a number of different PL interactions, including protein-carbohydrate, protein-fatty acid, and protein-small molecule complexes [23,47,50]. The origin of the stabilizing effects of imidazole is believed to be due, at least in part, to enhanced evaporative cooling resulting from the dissociation of nonspecific imidazole adducts from the gaseous PL ions [47].…”

“…The combination of competitive binding experiments and ESI-MS detection has been exploited in numerous studies to extract binding data that could not be measured directly by ESI-MS. For example, the range of K a values that can be measured by ESI-MS can be dramatically extended through competition experiments involving multiple proteins that exhibit a range of affinities for the same L [34,35]. More recently, it was demonstrated that the affinities of labile PL complexes that are prone to dissociation in the gas phase are readily determined by monitoring the interaction between the P of interest and a "reference" L, which binds competitively and forms a stable (in the gas phase) complex with P, in the presence of L [23,36].…”

Time to Face the Fats: What Can Mass Spectrometry Reveal about the Structure of Lipids and Their Interactions with Proteins?

“…In the case of SF-1, its affinity for a range of different phospholipids was explored by treating the protein with liposomes of different constitution and monitoring the abundance of the resulting adducts by ESI mass spectrometry [7]. More recently, Klassen and coworkers demonstrated that stable complexes between bovine β-lactoglobulin and different fatty acids could be formed by nano-electrospray ionization [49]. In this study, in-source dissociation of the delicate complexes was minimized by the addition of a high concentration of imidazole to the electrospray solution.…”