Quantifying Protein-Ligand Binding Constants using Electrospray Ionization Mass Spectrometry: A Systematic Binding Affinity Study of a Series of Hydrophobically Modified Trypsin Inhibitors

Abstract

NanoESI-MS is used for determining binding strengths of trypsin in complex with two different series of five congeneric inhibitors, whose binding affinity in solution depends on the size of the P3 substituent. The ligands of the first series contain a 4-amidinobenzylamide as P1 residue, and form a tight complex with trypsin. The inhibitors of the second series have a 2-aminomethyl-5-chloro-benzylamide as P1 group, and represent a model system for weak binders. The five different inhibitors of each group are based on the same scaffold and differ only in the length of the hydrophobic side chain of their P3 residue, which modulates the interactions in the S3/4 binding pocket of trypsin. The dissociation constants (K D ) for high affinity ligands investigated by nanoESI-MS ranges from 15 nM to 450 nM and decreases with larger hydrophobic P3 side chains. Collision-induced dissociation (CID) experiments of five trypsin and benzamidine-based complexes show a correlation between trends in K D and gas-phase stability. For the second inhibitor series we could show that the effect of imidazole, a small stabilizing additive, can avoid the dissociation of the complex ions and as a result increases the relative abundance of weakly bound complexes. Here the K D values ranging from 2.9 to 17.6 μM, some 1-2 orders of magnitude lower than the first series. For both ligand series, the dissociation constants (K D ) measured via nanoESI-MS were compared with kinetic inhibition constants (K i ) in solution.

Cis→Trans Isomerization of Pro7 in Oxytocin Regulates Zn2+ Binding

“…Many studies have been performed to determine binding constants for various protein–ligand complexes with ESI-MS [41–46]. In this study, we use IMS-MS data to calculate K A s for Zn 2+ bound to OT.…”

Determination of thermodynamic and kinetic properties of biomolecules by mass spectrometry

“…In this way they quantified the affinities of methylmannose polysaccharides for lipids [65]. Cubrilovic et al have looked at the binding strength of trypsin with different five congeneric inhibitors whose structure differs only in the substituent group and found that nano-ESI-MS could successfully capture the trend in the binding affinity [66]. The effect of DMSO, a common solvent in high-throughput screening studies, on protein-ligand binding equilibrium was also studied [67].…”

Origin and prediction of free-solution interaction studies performed label-free

“…However, back-scattering interferometry (BSI) is a free-solution label-free technique with the added benefit of sensitivity that rivals fluorescence (9). There are other techniques performed in free solution, such as MS (10, 11) and NMR (12,13) and the widely used isothermal titration calorimetry (ITC) (14, 15). As with NMR, ITC has many advantages, but exhibits modest sensitivity and often requires large sample quantities.…”