Screening for High-Affinity Ligands to theSrcSH2 Domain Using Capillary Isoelectric Focusing-Electrospray Ionization Ion Trap Mass Spectrometry

Abstract

A solution-based microscale approach for determination of high-affinity noncovalent complexes from mixtures of compounds is presented, based on capillary isoelectric focusing coupled on-line with electrospray ionization ion trap mass spectrometry. The studies are performed using the src homology 2 domain and tyrosine-phosphorylated peptide ligands as a model system. Tight complexes are formed in solution, preconcentrated up to 2 orders of magnitude and separated on the basis of their isoelectric points. The complexes are then dissociated in the mass spectrometer and the freed ligands identified. Picomole or less amounts of protein reagent are consumed per experiment. Structural information for the ligands involved in tight complex formation may be obtained using the MSn capabilities of the ion trap. The methodology can potentially be used to screen rapidly combinatorial mixtures of compounds for high-affinity ligands.

Capillary Isoelectric Focusing Separation Combined with Mass Spectrometry Using Sonic Spray Ionization for Protein Analysis.

“…However, they are not simple procedures; that is, they involve either of two types: after CIEF separation between the inlet and outlet reservoirs, one end of the separation capillary is removed from the reservoir and inserted into a sheath-flow interface 3), 5), 7)῍9) ; or the outlet catholyte is exchanged immediately for an acidic sheath liquid in the sheath-flow interface. 4) We have recently developed a sonic spray ionization (SSI) interface for capillary electrophoresis/mass spectrometry (CE/MS): it has a bu#er reservoir between the sample-introduction capillary of the ion source and the electrophoresis-separation capillary. 10) In SSI, a sample solution is sprayed from a sample-introduction capillary by a high-speed nitrogen-gas flow that is coaxial to the capillary, and ions of the chemicals in the solution, as well as charged droplets, are produced at room temperature and atmospheric pressure.…”

Monitoring enzyme reaction and screening of inhibitors of acetylcholinesterase by quantitative matrix-assisted laser desorption/ionization fourier transform mass spectrometry

“…The use of electrospray ionization (ESI)-MS is rapidly expanding into the study of enzyme kinetics [17][18][19][20][21][22][23]. Also, combinations of chromatographic and electrophoretic separation methods with ESI-MS, such as liquid chromatography (LC)/MS [24], LC/tandem mass spectroscopy (MS/MS) [25], gas chromatography (GC)/MS [26], frontal affinity chromatography (FAC)/MS [27], or capillary electrophoresis (CE)/MS [28] have been reported for the determination of acetylcholine or the screening of AChE inhibitors. These methods are quite specific but have longer cycle times.…”

Automated Nano-Electrospray Mass Spectrometry for Protein-Ligand Screening by Noncovalent Interaction Applied to Human H-FABP and A-FABP

“…FT-ICR/MS in combination with microelectrospray has been used for the simultaneous analysis of a 324-member peptide library screened against the Hck Src homology 2 domain receptor. 11 The combination of capillary isoelectric focusing-ESI/MS to screen high-affinity ligands against the Src SH2 domain by noncovalent interaction was shown by Lyubarskaya et al 12 These authors used online selection and online structural identification of strongly bound ligands. Greig and Robinson 13 described detection of oligonucleotide-ligand complexes by ESI-MS (DOLCE-MS) as a component of HTS.…”