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We describe an approach for the determination of binding constants for protein-ligand complexes with electrospray ionization mass spectrometry, based on the observation of unbound ligands competing for binding to a protein target. For the first time, dissociation constants lower than picomolar could be determined with good accuracy by electrospray ionization mass spectrometry. The presented methodology relies only on the determination of signal intensity ratios for free ligands in the low mass region. Therefore, all the advantages of measuring low masses with mass spectrometry, such as high resolution are preserved. By using a reference ligand with known binding affinity, the affinity of a second ligand can be determined. Since no noncovalently bound species are observed, assumptions about response factors are not necessary. The method is validated with ligands binding to avidin and applied to ligands binding to p38 mitogen-activated protein kinase.

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 (KD) of the model system hen egg white lysozyme (HEWL) binding to N,N',N''-triacetylchitotriose (NAG3). The best KD value compared with solution data were found for ESSI, 19.4 +/- 3.6 microM. Then, we determined the KDs of the two nucleotide binding sites of adenylate kinase (AK), where we obtained KDs of 2.2 +/- 0.8 microM for the first and 19.5 +/- 8.0 microM for the second binding site using ESSI. We found a weak charge state dependence of the KD for both protein-ligand systems, where for all ionization techniques the KD value decreases with increasing charge state. We demonstrate that ESSI is very gentle and insensitive to instrumental parameters, and the KD obtained is in good agreement with solution phase results from the literature. In addition, we tried to determine the KD 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 KD values with a strong charge state dependence, which were in no case close to literature values for solution phase.

Matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) of noncovalent protein complexes is difficult, due to the disruptive nature of processes occurring during MALDI sample preparation and ion formation. Sometimes the observation of intact noncovalent protein complexes with MALDI is only possible if data are acquired from the first laser shot fired at a fresh sample; this is called the 'first shot phenomenon'. To study the origin of the first shot phenomenon, we used MALDI-MS and confocal laser scanning microscopy (CLSM) to examine typical MALDI sample preparations with embedded protein complexes, labeled with fluorophores. Fluorescence energy transfer techniques allowed the differentiation between intact and dissociated protein complexes with CLSM. In cases where a first shot behavior was observed by MALDI-MS, it was found to be accompanied by localization of protein complexes at the exterior of the sample crystals. Segregation of the large protein complexes to the exterior and dissociation of the complexes in the crystal interior during sample crystallization can rationalize this observation.