Elena N. Kitova

Reliable determinations of protein-ligand interactions by direct ESI-MS measurements: Are we there yet?

The association-dissociation of noncovalent interactions between protein and ligands, such as other proteins, carbohydrates, lipids, DNA, or small molecules, are critical events in many biological processes. The discovery and characterization of these interactions is essential to a complete understanding of biochemical reactions and pathways and to the design of novel therapeutic agents that may be used to treat a variety of diseases and infections. Over the last 20 y, electrospray ionization mass spectrometry (ESI-MS) has emerged as a versatile tool for the identification and quantification of protein–ligand interactions in vitro. Here, we describe the implementation of the direct ESI-MS assay for the determination of protein–ligand binding stoichiometry and affinity. Additionally, we outline common sources of error encountered with these measurements and various strategies to overcome them. Finally, we comment on some of the outstanding challenges associated with the implementation of the assay and highlight new areas where direct ESI-MS measurements are expected to make significant contributions in the future.

Hydrophobic protein-ligand interactions preserved in the gas phase.

The results of time-resolved thermal dissociation measurements and molecular dynamic simulations are reported for gaseous deprotonated ions of the specific complexes of bovine beta-lactoglobulin (Lg) and a series of the fatty acids (FA): CH(3)(CH(2))(x)COOH, where x = 10, 12, 14, and 16. At the reaction temperatures investigated, 25-66 degrees C, the gaseous ions dissociate exclusively by the loss of neutral FA. According to the kinetic data, and confirmed by ion mobility measurements, the (Lg + FA)(7-) ions exist in two, noninterconverting structures designated the fast (Lg + FA)(f)(7-) and slow (Lg + FA)(s)(7-) components. The Arrhenius parameters for both components are sensitive to the length of the FA aliphatic chain. For the fast components, the activation energy (E(a)) increases in a nearly linear fashion, with each methylene group contributing approximately 0.8 kcal mol(-1) to E(a). This is similar to the contribution of -CH(2)- groups to the solvation of n-alkanes in nonpolar solvents. Furthermore, the magnitude of the E(a) values for the fast components is similar to the solvation enthalpies expected for the FA aliphatic chains in nonpolar and weakly polar solvents. The E(a) values determined for the slow components are larger than those of the fast components. Furthermore, the E(a) values do not vary in a simple fashion with the length of the aliphatic chain. Molecular dynamics simulations performed on the (Lg + PA) complex revealed that, depending on the charge configuration, the (Lg + PA)(7-) ion can exist in two distinct structures, which differ primarily by the position of the EF loop. In the open structure the EF loop is positioned away from the entrance to the hydrophobic cavity and the ligand is stabilized only through nonpolar intermolecular interactions. In the closed structure the EF loop covers the entrance of the cavity and the carboxylic group of PA participates in H-bonds with residues on the EF loop or residues located at the entrance of the cavity. The loss of ligand from the closed structure would require both the cleavage of the H-bonds and the nonpolar contacts. Taken together, these results suggest that the aliphatic chain of the FA remains bound within the hydrophobic cavity in the gas phase (Lg + FA)(7-) ions. Furthermore, the barrier to dissociation of the (Lg + FA)(f)(7-) ions reflects predominantly the cleavage of the nonpolar intermolecular interactions, while for the (Lg + FA)(s)(7-) ions the FA is stabilized by both nonpolar interactions and H-bonds.

Functional properties of the carboxy-terminal host cell-binding domains of the two toxins, TcdA and TcdB, expressed by Clostridium difficile

The biological and ligand-binding properties of recombinant C-terminal cell-binding domains (CBDs) and subdomains of the two large exotoxins, Toxin A (TcdA) and Toxin B (TcdB) expressed by Clostridium difficile were examined in the hemagglutination and Verocytotoxicity neutralization assays and by qualitative affinity chromatography using Sepharose-linked alpha Gal(1,3)betaGal(1,4)beta Glc as well as the direct electrospray ionization mass spectrometry (ES-MS) assay. These studies revealed that, whereas the full-length TcdA CBD agglutinated rabbit erythrocytes, neutralized TcdA-mediated Vero cell death and bound to alpha Gal(1,3)betaGal(1,4)beta Glc-derivatized Sepharose, the TcdB CBD was inactive in these functional assays. Moreover, retention by alpha Gal(1,3)betaGal(1,4)beta Glc-derivatized Sepharose corresponded to the number of available TcdA subdomain ligand-binding sites. By contrast, the ES-MS assays revealed that both the TcdA and TcdB CBD bind to 8-methoxycarbonyloctyl-alpha Gal(1,3)betaGal(1,4)beta Glc sequences with similar avidities. Additional ES-MS experiments using chemically altered alpha Gal(1,3)betaGal(1,4)beta Glc sequences also revealed that the TcdA and TcdB CBD will tolerate a fair amount of structural variation in their complementary glycan ligands. Although the studies are consistent with the known ligand-binding properties of the TcdA and TcdB holotoxins, they also revealed subtle heretofore unrecognized functional differences in their receptor recognition properties.

Gas Phase Stabilization of Noncovalent Protein Complexes Formed by Electrospray Ionization

The use of gas phase additives to stabilize noncovalent protein complexes in electrospray ionization mass spectrometry (ES-MS) is demonstrated for two protein-ligand interactions, an enzyme-small molecule inhibitor complex, and a protein-disaccharide complex. It is shown that the introduction of gas phase imidazole into the ES ion source effectively protects gas phase protein-ligand complexes against in-source dissociation. The stabilizing effect of imidazole vapor is comparable to that observed upon addition of imidazole to the ES solution. The introduction of sulfur hexafluoride, at high partial pressure, into the source region also effectively suppresses in-source dissociation of protein complexes. It is proposed that evaporative cooling is the primary mechanism responsible for the stabilizing effects observed for the gas phase additives.

Quantifying labile protein—Ligand interactions using electrospray ionization mass spectrometry

A new electrospray ionization mass spectrometry (ES-MS) approach for quantifying protein—ligand complexes that are prone to in-source (gas-phase) dissociation is described. The method, referred to here as the reference ligand ES-MS method, is based on the direct ES-MS assay and competitive ligand binding. A reference ligand (Lref), which binds specifically to the protein (P), at the same binding site as the ligand (L) of interest, with known affinity and forms a stable protein—ligand complex in the gas phase, is added to the solution. The fraction of P bound to Lref, which is determined directly from the ES mass spectrum, is sensitive to the fraction of P bound to L in solution and enables the affinity of P for L to be determined. A mathematical framework for the implementation of the method in cases where P has one or two specific ligand binding sites is given. Affinities of two carbohydrate-binding proteins, a single chain fragment of a monoclonal antibody and the lectin concanavalin A, for monosaccharide ligands are reported and the results are shown to agree with values obtained using isothermal titration calorimetry.

Quantifying Ligand Binding to Large Protein Complexes Using Electrospray Ionization Mass Spectrometry

An electrospray ionization mass spectrometry (ESI-MS) method for quantifying protein-ligand complexes that cannot be directly detected by ESI-MS is described. The proxy protein ESI-MS method combines direct ESI-MS binding measurements with competitive protein-ligand binding. To implement the method, a proxy protein (P(proxy)), which interacts specifically with the ligand of interest with known affinity and can be detected directly by ESI-MS, is used to quantitatively monitor the extent of ligand binding to the protein of interest. A mathematical framework for establishing the association constant (K(a)) for protein-ligand binding by the proxy protein ESI-MS method, implemented with a P(proxy) containing a single ligand binding site, is given. A modified form of the proxy protein ESI-MS method, which accounts for real-time changes in ligand concentration, is also described. The reliability of these methods is demonstrated for the interactions between the 180 kDa wildtype homotrimeric tailspike protein of the bacteriophage P22 and its endorhamnosidase point mutant (D392N) with its ligands comprising two and three O-antigen repeats from Salmonella enterica serovar Typhimurium: octasaccharide ([α-Gal-(1→2)-[α-Abe-(1→3)]-α-Man-(1→4)-α-Rha](2)) and dodecasaccharide ([α-Gal-(1→2)-[α-Abe-(1→3)]-α-Man-(1→4)-α-Rha](3)). A 27 kDa single chain antibody, which binds to both ligands, served as P(proxy). The results of binding measurements performed at 10 and 25 °C are in excellent agreement with K(a) values measured previously using a fluorescence quenching assay.

Protein–Glycosphingolipid Interactions Revealed Using Catch-and-Release Mass Spectrometry

Glycosphingolipids (GSL) on the surface of cells are important receptors in antigen/microbial recognition and cell adhesion. However, their functional characterization is often challenging. We have developed a catch-and-release electrospray ionization mass spectrometry (CaR-ESI-MS) assay for the identification of specific interactions between water-soluble proteins or protein complexes with GSL incorporated into nanodiscs. The specificity and sensitivity of the assay is demonstrated for interactions involving cholera toxin and Shiga toxin, with their natural GSL receptors, the ganglioside GM1, and the globotriaosylceramide Gb3, respectively. The detection of binding between cholera toxin and GM1 within a mixture of lipids extracted from cell membranes highlights the potential of this assay for the discovery of biologically relevant protein-GSL interactions.

Thermal dissociation of the protein homodimer ecotin in the gas phase

The influence of charge on the thermal dissociation of gaseous, protonated, homodimeric, protein ecotin ions produced by nanoflow electrospray ionization (nanoES) was investigated using the blackbody infrared radiative dissociation technique. Dissociation of the protonated dimer, (E2 + nH)n+≡ E2n+ where n = 14–17, into pairs of monomer ions is the dominant reaction at temperatures from 126 to 175 °C. The monomer pair corresponding to the most symmetric charge distribution is preferred, although 50–60% of the monomer product ions correspond to an asymmetric partitioning of charge. The relative abundance of the different monomer ion pairs produced from E214+, E215+, and E216+ depends on reaction time, with the more symmetric charge distribution pair dominating at longer times. The relative yield of monomer ions observed late in the reaction is independent of temperature indicating that proton transfer between the monomers does not occur during dissociation and that the different monomer ion pairs are formed from dimer ions which differ in the distribution of charge between the monomers. For E217+, the yield of monomer ions is independent of reaction time but does exhibit slight temperature dependence, with higher temperatures favoring the monomers corresponding to most symmetric charge distribution. The charge distribution in the E215+ and E216+ dimer ions influences the dissociation kinetics, with the more asymmetric distribution resulting in greater reactivity. In contrast, the charge distribution has no measurable effect on the dissociation kinetics and energetics of the E217+ dimer.

Nonspecific interactions between proteins and charged biomolecules in electrospray ionization mass spectrometry

An investigation of the nonspecific association of small charged biomolecules and proteins in electrospray ionization mass spectrometry (ES-MS) is described. Aqueous solutions containing pairs of proteins and a small acidic or basic biomolecule that does not interact specifically with either of the proteins were analyzed by ES-MS and the distributions of the biomolecules bound nonspecifically to each pair of proteins compared. For the basic amino acid arginine and the peptide RGVFRR, nonequivalent distributions were measured in positive ion mode, but equivalent distributions were measured in negative ion mode. In the case of uridine 5′-diphosphate, nonequivalent distributions were measured in negative ion mode, but equivalent distributions observed in positive ion mode. The results of dissociation experiments performed on the gaseous ions of the nonspecific complexes suggest that the nonequivalent distributions result from differences in the extent to which the nonspecific complexes undergo in-source dissociation. To test this hypothesis, the distributions of nonspecifically bound basic molecules measured in the presence of imidazole, which protects complexes from in-source dissociation, were compared. In all cases, equivalent distributions were obtained. The results indicate that nonspecific binding of charged molecules to proteins during ES is a statistical process, independent of protein structure and size. However, the kinetic stabilities of the nonspecific interactions are sensitive to the nature of the protein ions. It is concluded that the reference protein method for correcting ES mass spectra for nonspecific ligand-protein binding can be applied to the analysis of ionic ligands, provided that in-source dissociation of the nonspecific interactions is minimized.

Elucidating the intermolecular interactions within a desolvated protein-ligand complex. An experimental and computational study.

The first detailed study of the intermolecular hydrogens bonds (H-bonds) within a desolvated, noncovalent protein-ligand complex is reported. Using both experimental and computational methods, the intermolecular H-bonds stabilizing protonated and deprotonated ions of a complex composed of a single chain fragment (scFv) of a monoclonal antibody and its native trisaccharide ligand, alphaGal[alphaAbe] alphaMan (1), are characterized. Using the blackbody infrared radiative dissociation-functional group replacement (BIRD/FGR) technique, three H-bond donor-acceptor pairs within the gaseous (scFv + 1)n+ ions are identified and quantified. Additional sites of interaction on the protein and ligand, for which the binding partner could not be elucidated, are also identified. Comparison of the gas-phase interaction maps with the crystal structure suggests that at least two of the specific H-bonds are conserved upon transfer of the complex from solution to the gas phase by electrospray ionization. However, new (nonspecific) interactions can also form in the gas phase. Notably, the nature and strength of the intermolecular interactions can vary significantly with charge state, and striking differences in the structures of the (scFv + 1)n+ and (scFv + 1)n- ions are evident. Intermolecular H-bonds are also identified from molecular dynamics (MD) simulations performed at the +8 and -8 charge states. Agreement is found for a majority of intermolecular interactions predicted for the (scFv + 1)8+ ion by the MD simulation and BIRD/FGR method; the agreement is less favorable in the case of the (scFv + 1)8- ion. However, both the computational and experimental results point to structural differences between the +8 and -8 ions. The computational results also provide insights into the structural changes that accompany the loss of interfacial waters from the complex.