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.
Subject(s)
Peptides/analysis , Phosphotyrosine/analysis , Electrophoresis, Capillary , Isoelectric Focusing , Mass Spectrometry , Spectrophotometry, UltravioletABSTRACT
On-line affinity capillary electrophoresis-electrospray ionization-mass spectrometry (ACE-MS) was used for the simultaneous measurement of multiple binding constants of an all-D-tetrapeptide library to the model receptor, vancomycin. Determination of Kd values for the 19 peptides of the form Fmoc-DXYA is demonstrated. The data are compared with the results obtained for individual compounds using ACE-UV, and good correlation between the two detection methods is shown. Simultaneous determination of multiple Kd values by ACE-MS is achieved in one set of experiments, whereas only one Kd value can be obtained by ACE-UV during the same time. ACE-MS measures multiple binding constants in solution in a fast and reliable manner using femtomole amounts of samples.
Subject(s)
Peptides/chemistry , Vancomycin/chemistry , Binding, Competitive , Electrophoresis, Capillary/methods , Mass Spectrometry/methods , Models, ChemicalABSTRACT
The potential of a new microanalytical method using magnetic beads (MBs) and commercial capillary electrophoresis (CE) instrumentation for performing enzymatic and inhibition assays, as well as for analysis of biological molecules such as antigens, substrates, etc., has been explored. A small quantity of magnetic beads containing immobilized biomolecules was injected into a neutral hydrophilic-coated fused-silica capillary. The short plug (2-3 mm) of beads was held fixed by a magnet placed in the cartridge of the CE system, without the use of frits. The beads could be replaced after each run, eliminating the need to regenerate the solid support. Two protocols were used for analysis: sequential injection (SI) and SI followed by isotachophoretic (ITP) focusing. Alkaline phosphatase (AP) and HIV-protease were used to demonstrate the SI procedure for enzymatic and inhibition assays. The second protocol, SI/ITP, was employed to quantitate an antigen (mouse mAB) using antibodies (sheep IgG towards mouse AB) immobilized on the beads. The MB-CE method, requiring only femtomole (fmol) quantities of material, can potentially be employed in diagnostic and forensic assays, kinetic studies and searching for inhibitors, ligands, receptors, etc.
Subject(s)
Alkaline Phosphatase/analysis , Electrophoresis, Capillary/methods , Enzymes, Immobilized/analysis , HIV Protease/analysis , Alkaline Phosphatase/chemistry , Alkaline Phosphatase/immunology , Animals , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/immunology , Enzymes, Immobilized/chemistry , Enzymes, Immobilized/immunology , HIV Protease/chemistry , HIV Protease Inhibitors/chemistry , Magnetics , Mice , Microspheres , Osmolar Concentration , Pepstatins/chemistry , Spectrometry, Fluorescence , Time FactorsABSTRACT
Using beta-endorphin as a model system, a new microscale solution-based approach for linear epitope mapping based on affinity capillary electrophoresis-mass spectrometry (ACE-MS) is demonstrated. Tryptic peptides are separated in a neutral coated capillary and monitored by ultraviolet absorbance and electrospray mass spectrometry. Then, following injection of the peptide digest mixture, anti-beta-endorphin antibody is injected. The peptide, which binds to the antibody, is captured and disappears from its migration time. Following this subtraction-screening procedure, the binding of the individually synthesized or isolated immunoreactive peptide is examined by the ACE-MS procedure to confirm that the epitope resides on the peptide. A series of truncated peptides can then be made and the precise epitope determined by ACE-MS. The method utilizes low femtomole amounts of antibody and peptide digest per run and is rapid and easily automatable.
