Diffusion measurements by electrospray mass spectrometry for studying solution-phase noncovalent interactions

Abstract

This study describes a novel approach for monitoring noncovalent interactions in solution by electrospray mass spectrometry (ESI-MS). The technique is based on measurements of analyte diffusion in solution. Diffusion coefficients of a target macromolecule and a potential low molecular weight binding partner are determined by measuring the spread of an initially sharp boundary between two solutions of different concentration in a laminar flow tube (Taylor dispersion), as described in Rapid Commun. Mass Spectrom. 2002Spectrom. , 16, 1454Spectrom. -1462. In the absence of noncovalent interactions, the measured ESI-MS dispersion profiles are expected to show a gradual transition for the macromolecule and a steep transition for the low molecular weight compound. However, if the two analytes form a noncovalent complex in solution the dispersion profiles of the two species will be very similar, since the translational diffusion of the small compound is determined by the slow Brownian motion of the macromolecule. In contrast to conventional ESI-MS-based techniques for studying noncovalent complexes, this approach does not rely on the preservation of solution-phase interactions in the gas phase. On the contrary, "harsh" conditions at the ion source are required to disrupt any potential gasphase interactions between the two species, such that their dispersion profiles can be monitored separately. The viability of this technique is demonstrated in studies on noncovalent heme-protein interactions in myoglobin. Tight noncovalent binding is observed in solutions of pH 10, both in the absence and in the presence of 30% acetonitrile. In contrast, a significant disruption of the noncovalent interactions is seen at an acetonitrile content of 50%. Under these conditions, the diffusion coefficient of heme in the presence of myoglobin is only slightly lower than that of heme in a protein-free solution. A breakdown of the noncovalent interactions is also observed in aqueous solution of pH 2.4, where myoglobin is known to adopt an acid-unfolded conformation. (J Am Soc Mass Spectrom 2003, 14, 430 -441)

Controlled band dispersion for quantitative binding determination and analysis with electrospray ionization‐mass spectrometry

“…In a subsequent study that used the diffusion measurement, the noncovalent complex of heme with apo‐myoglobin was studied (Clark & Konermann, 2003). In the native form of myoglobin, the heme is bound very strongly by multiple interactions to the protein, to produce a K d on the order of 10 −14 M (Hargrove, Wilkinson, & Olson, 1996).…”

“…The diffusion coefficients determined from these fits, in units of 10 −10 m 2 sec −1 , are indicated in each panel. Reproduced with permission from Elsevier Science (Clark & Konermann, 2003) (Copyright 2003).…”

“…The purpose of this review is to highlight a few new and promising methods for on‐line quantitative binding determinations. ESI‐MS‐based diffusion measurements, introduced by the Konermann group (Clark, Leaist & Konermann, 2002; Clark & Konermann, 2003, 2004a,b), and dynamic titration measurements, introduced by the Schug group (Frycak & Schug, 2007, 2008), provide examples of how controlled band dispersion can be used to create well‐characterized temporal compositional analyte gradients from which quantitative binding information can be obtained. These methods increase the repertoire of available techniques to study solution‐phase interaction systems, particularly to facilitate the use of ESI‐MS in quantitative screening applications.…”

Practical Investigation of Molecular and Biomolecular Noncovalent Recognition Processes in Solution by ESI‐MS

“…Other approaches, such as those based on solution-phase H/D exchange (PLIM-STEX [98], SUPREX [99,100], and others [101]) and diffusion-based measurements [102,103] represent promising experimental set-ups for studying a wide range of molecular and macromolecular interaction systems. These are indirect methods based on the definition given previously and rely on detecting the dissociated receptor-ligand interaction partners through the use of either solution (e.g., for quenching of H/D exchange) or ionization conditions not amenable for detecting noncovalent complexes.…”

Recent developments in electrospray ionisation mass spectrometry: noncovalently bound protein complexes

“…A screening method for the identification of high-affinity ligands for specific macromolecular protein complexes by ESI-MS-based diffusion measurements has been reported. [52][53][54] The method relies on the postulation that noncovalent ligandcomplex interactions will reduce the diffusion coefficient of the ligand and the interaction between lysozyme and a number of polysaccharides has been studied. 53,54 Data interpretation of spectra obtained from macromolecular protein complexes is often performed manually as the currently available deconvolution algorithms 55, 56 which work well in converting the wide charge state distribution of an ionised denatured protein into a single peak on a zero charge, or mass, scale, do not always cope well with charge state distributions which cover only a small number of integer values, or with mixtures of oligomers based on the same monomeric protein unit.…”