Kinetic Size-Exclusion Chromatography with Mass Spectrometry Detection: An Approach for Solution-Based Label-Free Kinetic Analysis of Protein–Small Molecule Interactions

Abstract

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 (KSEC-MS), 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.

Analysis of Hormone–Protein Binding in Solution by Ultrafast Affinity Extraction: Interactions of Testosterone with Human Serum Albumin and Sex Hormone Binding Globulin

“…29,30,36,37 Only 1–50 μL of sample and as little as 1.25 pmol testosterone, 0.5 pmol SHBG, or 20 pmol HSA were needed per injection, which is less than the amounts of sample that have been used in many prior methods for such work. 7,9,14,18,24 This feature was particularly useful in experiments that involved a relatively expensive binding agent such as SHBG. This approach only required peak area measurements and the use of a linear fit for a first-order dissociation process, which made the data analysis easier to carry out than in other recent dissociation-based methods (e.g., those based on CE or size-exclusion chromatography).…”

“…This approach only required peak area measurements and the use of a linear fit for a first-order dissociation process, which made the data analysis easier to carry out than in other recent dissociation-based methods (e.g., those based on CE or size-exclusion chromatography). 24,25 In addition, this technique could measure free hormone fractions in less than 6–9 min, as opposed to analysis times ranging from 22 min to several hours in prior methods used to examine testosterone-protein interactions or related systems. 7,9,14,18–20,23,24 The combined use of the single column and two-dimensional systems also made it possible to significantly increase the range of solute concentrations, rate constants and equilibrium constants that could be examined by ultrafast affinity extraction (e.g., detection at low nM levels, k d values spanning from at least 10 −2 to 10 s −1 , and K a or nK a ′ values ranging from at least 10 4 to 10 9 M −1 ).…”

Moderate to high throughput in vitro binding kinetics for drug discovery

“…High throughput kinetic profiling of enzyme inhibitors/activators or receptor antagonists/agonists by global progress curve analysis has been developed (40,41). The kinetic size-exclusion chromatography method is able to monitor kinetics on the timescale of seconds to minutes (42,43). For slow timescale kinetics that takes hours to days, preincubation followed by jump dilution is used to monitor the very slow dissociation of a binary complex (44).…”

Capillary Electrophoresis for Quantitative Studies of Biomolecular Interactions

“…It could expand the arsenal of available separation modes while potentially adopting data processing approaches similar to those developed for KCE. 113 In this review, we have tried to clarify the nomenclature for KCE methods and invite a wider research community to using this approach. In our view, further progress of the KCE platform should involve: (i) methodological development of the KCE approach; (ii) detailed study of advanced computational models and building new mathematics with userfriendly solutions, thus, to fill the gaps in Table 1; (iii) enhancement of technical tools for the KCE methods; (iv) miniaturization of KCE technical basis; (v) application of KCE methods to new ligand-target systems; (vi) validation of KCE methods; (vii) hyphenation of KCE with other instrumental platforms to realize multidimensional analysis and accelerate analytical capabilities of KCE-based methods.…”