Which Electrospray-Based Ionization Method Best Reflects Protein-Ligand Interactions Found in Solution? A Comparison of ESI, nanoESI, and ESSI for the Determination of Dissociation Constants with Mass Spectrometry

We present a comparison of three different electrospray-based ionization techniques for the investigation of noncovalent complexes with mass spectrometry. The features and characteristics of standard electrospray ionization (ESI), chip-based nanoESI, and electrosonic spray ionization (ESSI) mounted onto a hybrid quadrupole time-of-flight mass spectrometer were compared in their performance to determine the dissociation constant ( K D) of the model system hen egg white lysozyme (HEWL) binding to N, N′, N″-triacetylchitotriose (NAG 3). The best K D value compared with solution data were found for ESSI, 19.4 ± 3.6 μM. Then, we determined the K Ds of the two nucleotide binding sites of adenylate kinase (AK), where we obtained K Ds of 2.2 ± 0.8 μM for the first and 19.5 ± 8.0 μM for the second binding site using ESSI. We found a weak charge state dependence of the K D for both protein-ligand systems, where for all ionization techniques the K D value decreases with increasing charge state. We demonstrate that ESSI is very gentle and insensitive to instrumental parameters, and the K D obtained is in good agreement with solution phase results from the literature. In addition, we tried to determine the K D for the lymphocyte-specific kinase LCK binding to a kinase inhibitor using nanoESI due to the very low amount of sample available. In this case, we found K D values with a strong charge state dependence, which were in no case close to literature values for solution phase.