Determination of dissociation constants via quantitative mass spectrometry

Abstract

The interplay of biomolecules governs all cellular processes. Qualitative analysis of such interactions between biomolecules as well as the quantitative assessment of their binding affinities are essential for the understanding of biochemical mechanisms. As scientific interest therefore moves beyond pure structural investigation, methods that allow for the investigation of such interactions become increasingly relevant. In this perspective we outline classical methods that are applicable for the determination of binding constants and highlight specifically mass spectrometry based methods. The use of mass spectrometry to gain quantitative information about binding affinities however is a still developing field. Here, we discuss different approaches, which emerged over the last years to determine dissociation constants (KD) with mass spectrometry based methods. Specifically, we highlight the recent development of quantitative Laser Induced Liquid Bead Ion Desorption (qLILBID) mass spectrometry for the example of double stranded deoxyribonucleic acids as well as for different RNA—RNA binding protein systems. We show that quantitative laser induced liquid bead ion desorption can successfully be used for the top down investigation of complexes and their dissociation constants values ranging from low nM to low µM affinities.

ABC of Kd Accuracy

“…The necessity of appropriate calibration for signal-detecting instruments is well-known, and the calibration procedures vary from instrument to instrument. [59][60][61] In Supporting Information (Note S7 and Figure S2), we demonstrate the effect of a mis-calibrated fluorescence detector on Kd,det.…”

“…Horizontally polarized excitation light ensures that the fluorophores' exited-state distribution is along the detection axis, thereby equalizing the intensities of emitted light with different polarization orientations on the plane of detection, which is perpendicular to the travel direction of the emitted light. 59,60 In this scenario, any difference in the detected intensities of vertically and horizontally polarized emissions is caused by instrumental bias, which can be corrected by the determined G factor. Since G is dependent on wavelength and instrumental setup, 34, 59 its value should be re-determined when any optical component, e.g., emission filter and excitation/emission polarizer, is changed in the instrument.…”

Determination of the microscopic acid dissociation constant of piperacillin and identification of dissociated molecular forms

“…Mass spectrometry is used for resolving degradation/dissociation products ( Ahmed et al, 2012 ; Liang et al, 2017 ) and noncovalent interactions ( Kempen and Brodbelt, 2000 ; Hardouin and Lange, 2005 ; Zhang et al, 2006 ; Erba and Zenobi, 2011 ). A recent review reported its potential for the determination of dissociation constants and giving information about the specificity of noncovalent interactions ( Schulte et al, 2023 ). Several reports have presented evidence when properly controlled experimental conditions are used, electrospray ionization mass spectrometry (ESI-MS) has demonstrated its use in the detection and study of weakly bound forms.…”