Affinity classification of kinase inhibitors by mass spectrometric methods and validation using standard IC(50) measurements.

Protein kinases have emerged as a major drug target in the last years. Since more than 500 kinases are encoded in the human genome, cross-reactivity of a majority of kinase inhibitors causes problems. Tools are required for a rapid classification of inhibitors according to their affinity for a certain target to refine the search for new, more specific lead compounds. Mass spectrometry (MS) is increasingly used in pharmaceutical research and drug discovery to investigate protein-ligand interactions and determination of binding affinities. We present a comparison of different existing nanoelectrospray-MS based methods to quantify binding affinities and qualitatively rank, by competitive experiments, the affinity of several clinical inhibitors. We also present a new competitive method which is derived from our previous work for quantitative assessment of binding strengths (Wortmann et al., J. Mass Spectrom. 2008, 43(5), 600-608). The human kinases studied for this purpose were p38alpha (MAPK14) and LCK (lymphocyte specific kinase), and their interaction with 17 known small molecule kinase inhibitors was probed. Moreover, we present a new method to differentiate type I from type II inhibitors (Liu, Y.; Gray, N. S. Nat. Chem. Biol. 2006, 2(7), 358-364) based on a kinetic experiment with direct MS read-out of the noncovalent complex between the human kinase and the inhibitor. This method was successfully applied to p38alpha binding to BIRB796, as well as to a BIRB796 analogue. Quantitative determination of the binding strength is also described. The results of our competitive experiments for the affinity classification of different inhibitors, as well as the results for the kinetic study, are in good agreement with IC(50) measurements and data found in the literature.

Atmospheric pressure glow discharge desorption mass spectrometry for rapid screening of pesticides in food.

Flowing afterglow atmospheric pressure glow discharge tandem mass spectrometry (APGD-MS/MS) is used for the analysis of trace amounts of pesticides in fruit juices and on fruit peel. The APGD source was rebuilt after Andrade et al. (Andrade et al., Anal. Chem. 2008; 80: 2646-2653; 2654-2663) and mounted onto a hybrid quadrupole time-of-flight mass spectrometer. Apple, cranberry, grape and orange juices as well as fruit peel and salad leaves were spiked with aqueous solutions containing trace amounts of the pesticides alachlor, atrazine, carbendazim, carbofuran, dinoseb, isoproturon, metolachlor, metolcarb, propoxur and simazine. Best limits of determination (LODs) of pesticides in the fruit juices were achieved for metolcarb (1 microg/L in apple juice), carbofuran and dinoseb (2 microg/L in apple juice); for the analysis of apple skin best LODs were 10 pg/cm(2) of atrazine, metolcarb and propoxur which corresponds to an estimated concentration of 0.01 microg/kg apple, taking into account the surface area and the weight of the apple. The measured LODs were within or below the allowed maximum residue levels (MRLs) decreed by the European Union (1-500 microg/kg for pesticides in fruit juice and 0.01-5 microg/kg for apple skin). No sample pretreatment (extraction, pre-concentration, chromatographic separation) was necessary to analyze these pesticides by direct desorption/ionization using APGD-MS and to identify them using MS/MS. This makes APGD-MS a powerful high-throughput tool for the investigation of very low amounts of pesticides in fruit juices and on fruit peel/vegetable skin.

Ion internal energy distributions validate the charge residue model for small molecule ion formation by spray methods.

This paper reports a detailed study of the internal energy distribution of ions formed by four electrospray ionization (ESI)-related ionization methods, with particular emphasis on electrosonic spray ionization (ESSI). Substituted benzylpyridinium ions were used as thermometer ions to probe the internal energy distribution. The influence of different instrumental parameters was studied. Cone and skimmer voltages as well as the collision energy were found to strongly affect the ion internal energy distribution, whereas the distance between the emitter and the inlet of the mass spectrometer, the nebulizing gas pressure or the flow rate showed no influence. The internal energy distribution obtained with an ESSI source was compared with those obtained for electrospray (ESI), nanoelectrospray (nanoESI) and sonic spray ionization (SSI) on the same mass spectrometer with the same instrumental parameters. No clear differences were observed. As the charge residue model is the only ion formation mechanism possible for SSI, we conclude that benzylpyridinium ions are formed by the pathway suggested by this model.

Investigation of deprotonation reactions on globular and denatured proteins at atmospheric pressure by ESSI-MS.

Deprotonation reactions of multiply charged protein ions have been studied by introducing volatile reference bases at atmospheric pressure between an electrosonic spray ionization (ESSI) source and the inlet of a mass spectrometer. Apparent gas-phase basicities (GB(app)) of different charge states of protein ions were determined by a bracketing approach. The results obtained depend on the conformation of the protein ions in the gas phase, which is linked to the type of buffer used (denaturing or nondenaturing). In nondenaturing buffer, the GB(app) values are consistent with values predicted by the group of Kebarle using an electrostatic model (J. Mass Spectrom.2002, 38, 618) based on the crystal structures, but taking into account salt bridges between ionized basic and acidic sites on the protein surface. A new basicity order for the most basic sites was therefore obtained. An excellent agreement with the charge residue model (CRM) is obtained when comparing the observed and calculated maximum charge state. Decharging of the proteins in the electrosonic spray process could be also useful in the study on noncovalent complexes, by decreasing repulsive electrostatic interactions. A unified mechanism of the ESSI process is proposed.

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 (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.

Manipulation of charge states of biopolymer ions by atmospheric pressure ion/molecule reactions implemented in an extractive electrospray ionization source.

A home-made extractive electrospray ionization source is coupled to an linear quadrupole ion trap mass spectrometer to investigate ion/molecule reactions of biopolymers at ambient pressure. Multiply charged biopolymers such as peptides and proteins generated in an electrospray are easily reduced to a low charge state by the atmospheric pressure ion/molecule reactions occurring between the multiply charged ions and a strong basic reagent sprayed in neutral form into the electrospray plume. The charge state of the biopolymer ions can be manipulated by controlling the amount of the basic reagent. The production of biopolymer ions with low charge states results in a substantial improvement of sensitivity and reduced spectral congestion in ESI-MS. This is of importance for biopolymer mixture analysis and could have promising applications in proteomics.