The quest to develop even-simpler platforms for interrogation of protein–small molecule interactions continues. Despite dramatic advances in surface plasmon resonance and related techniques, the need still remains for approaches that do not require surface immobilization of proteins because this step has remained the most difficult aspect of such experiments. Here, the authors* provide a simple chromatography-based technique, coupled with novel data analysis, which allows the derivation of binding kinetics of small molecules interacting with proteins. In size-exclusion chromatography, an unbound small molecule travels slowly, eluting from the column last (together with salts, dyes, and/or other small molecule buffer components of the injected sample), while a fully bound ligand is expected to elute relatively fast as a part of its complex with the protein of interest (exact elution time of the latter depending on the molecular weight of the protein). The authors* recognized that between these extreme cases lies the typical situation in which given certain kon and koff of the complex, during migration through the size-exclusion column, the small molecule is likely to dissociate from the complex gradually and to begin to lag behind the protein, thus creating a specific profile of its concentration as a function of elution volume. The team used mass spectrometry for detection and quantitation of the small molecule in order to construct the profile (see figure) and then applied a series of equations to derive the kinetics of interaction. Data obtained from this method, which entails only injecting the complex and following the small molecule through mass spectrometry detector, compared favorably with results obtained through traditional means. The extreme simplicity of this new approach should make it an attractive alternative, especially in cases where the protein to be immobilized displays extreme sensitivity to such treatment.
* Abstract from Anal Chem 2014, Vol. 86:10016–10020
Studying the kinetics of reversible protein–small molecule binding is a major challenge. The available approaches require that either the small molecule or the protein be modified by labeling or immobilization on a surface. Not only can such modifications be difficult to do but also they can drastically affect the kinetic parameters of the interaction. To solve this problem, we present kinetic size-exclusion chromatography with mass spectrometry detection (KSECMS), a solution-based label-free approach. KSEC-MS utilizes the ability of size-exclusion chromatography (SEC) to separate any small molecule from any protein–small molecule complex without immobilization and the ability of mass spectrometry (MS) to detect a small molecule without a label. The rate constants of complex formation and dissociation are deconvoluted from the temporal pattern of small molecule elution measured with MS at the exit from the SEC column. This work describes the concept of KSEC-MS and proves it in principle by measuring the rate constants of interaction between carbonic anhydrase and acetazolamide.