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.

The blackbody infrared radiative dissociation technique was used to study the thermal decomposition of the gaseous B5 pentamer of the Shiga-like toxin I and its complexes with the Pk trisaccharide and a decavalent Pk-based oligosaccharide ligand (STARFISH, S). Dissociation of the protonated pentamer, (B5 + nH)n+ triple bond B5n+ where n = 11-14, proceeds almost exclusively by the loss of a single subunit (B) with a disproportionately large fraction (30-50%) of the parent ion charge. The degree of charge enrichment of the leaving subunit increases with increasing parent ion charge state. For n = 12-14, a distribution of product ion charge states is observed. The yields of the complementary pairs of product ions are sensitive to the reaction temperature, with higher temperatures favoring greater charge enrichment of the leaving subunit for +13 and +14, and the opposite effect for +12. These results indicate that some of the protons are rapidly exchanged between subunits in the gas phase. Dissociation of B5(14+) x S proceeds exclusively by the loss of one subunit, although the ligand increases the stability of the complex and also reduces the degree of charge enrichment in the ejected monomer. For B5(12+)(Pk)1-3, the loss of neutral Pk competes with loss of a subunit at low temperatures. Linear Arrhenius plots were obtained from the temperature-dependent dissociation rate constants measured for the loss of B from B5n+ and B514+ x S. The magnitude of the Arrhenius parameters is highly dependent on the charge state of the pentamer: Ea = 35 kcal/mol and A = 1,019 s(-1) (+14), 46 kcal/mol and 1,023 S(-1) (+13), 50 kcal/mol and 1026 s(-1) (+12), and 80 kcal/mol and 10(39) (+11). The Ea and A for B5(14+) x S are 59 kcal/mol and 10(30) s(-1), respectively. The reaction pathways leading to greater charge enrichment of the subunit lost from the B5(14+) and B5(13+) ions correspond to higher energy processes, however, these pathways are kinetically preferred at higher temperatures due to their large A factors. A simple electrostatic model, whereby charge enrichment leads to Coulombic repulsion-induced denaturation of the subunits and disruption of the intersubunit interactions, provides an explanation for the magnitude of the Arrhenius parameters and the origin of the asymmetric dissociation behavior of the complexes.

A new methodology for distinguishing between specific and nonspecific protein-ligand complexes in nanoelectrospray ionization mass spectrometry (nanoES-MS) is described. The method involves the addition of an appropriate reference protein (P(ref)), which does not bind specifically to any of the solution components, to the nanoES solution containing the protein(s) and ligand(s) of interest. The occurrence of nonspecific protein-ligand binding is monitored by the appearance of nonspecific (P(ref) + ligand) complexes in the nanoES mass spectrum. Furthermore, the fraction of P(ref) undergoing nonspecific ligand binding provides a quantitative measure of the contribution of nonspecific binding to the measured intensities of protein and specific protein-ligand complexes. As a result, errors introduced into protein-ligand association constants, K(assoc), as determined with nanoES-MS, by nonspecific ligand binding can be corrected. The principal assumptions on which this methodology is based, namely, that the fraction of proteins and protein complexes that engage in nonspecific ligand binding during the nanoES process is determined by the number of free ligand molecules in the offspring droplets leading to gaseous ions and is independent of the size and structure of the protein or protein complex, are shown to be generally valid. The application of the method for the determination of K(assoc) for two protein-carbohydrate complexes, under conditions where nonspecific ligand binding is prevalent, is demonstrated.

The influence of solution pH, analyte concentration and in-source dissociation on the measurement of the association constant for a single chain variable fragment of a monoclonal antibody (scFv) and its native trisaccharide ligand by nanoelectrospray-Fourier transform ion cyclotron resonance mass spectrometery has been systematically investigated. From the results of this study, experimental conditions that preserve the original distribution of bound and unbound protein in solution into the gas phase, such that the nanoES mass spectrum provides a quantitative measure of the solution composition, were identified. These include the use of short spray durations (<10 min) to minimize pH changes, equimolar concentrations of protein and ligand to minimize the formation of nonspecific complexes, and short accumulation times (<2 s) in the hexapole of the ion source to avoid collisional heating and dissociation of the gaseous complex. Application of this methodology to the scFv and a series of carbohydrate ligands yields results that are in agreement with values previously determined by isothermal titration calorimetry. Competitive binding experiments performed on solutions containing the scFv and a mixture of carbohydrate ligands were also found to yield accurate association constants.