Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:1.5.1.3 (dihydrofolate reductase)
 5,819 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Frontal affinity chromatography (FAC) interfaced with electrospray mass spectrometry (ESI-MS) has been reported as a potential method for screening of compound mixtures against immobilized target proteins. However, the interfacing of bioaffinity columns to ESI-MS requires that the eluent that passes through the protein-loaded column have a relatively low ionic strength to produce a stable spray. Such low ionic strength solvents can cause serious problems with protein stability and may also affect binding constants and lead to high nonspecific binding to the column. Herein, we report on the interfacing of bioaffinity columns to matrix-assisted laser desorption/ionization (MALDI) MS/MS as a new platform for FAC/MS studies. Capillary columns containing a monolithic silica material with entrapped dihydrofolate reductase were used for frontal affinity chromatography of small-molecule mixtures. The output from the column was combined with a second stream containing alpha-cyano-hydoxycinnamic acid in methanol and was deposited using a nebulizer-assisted electrospray method onto a conventional MALDI plate that moved relative to the column via a computer-controlled x-y stage, creating a semipermanent record of the FAC run. The use of MALDI MS/MS allowed for buffers with significantly higher ionic strength to be used for FAC studies, which reduced nonspecific binding of ionic compounds and allowed for better retention of protein activity over multiple runs. Following deposition, MALDI analysis required only a fraction of the chromatographic run time, and the deposited track could be rerun multiple times to optimize ionization parameters and allow signal averaging to improve the signal-to-noise ratio. Furthermore, high levels of potential inhibitors could be detected via MALDI with limited ion suppression effects. Both MALDI- and ESI-based analysis showed similar retention of inhibitors present in compound mixtures when using identical ionic strength conditions. The results show that FAC/MALDI-MS should provide advantages over FAC/ESI-MS for high-throughput screening of compound mixtures.
 ...
PMID:Capillary-scale frontal affinity chromatography/MALDI tandem mass spectrometry using protein-doped monolithic silica columns. 1588 27

Pathogenic Escherichia coli, one of the primary causes of urinary tract infections, has shown significant resistance to the most popular antibiotic, trimethoprim (TMP), which inhibits dihydrofolate reductase (DHFR). The resistance is modulated by single point mutations of DHFR. The impact of two clinically relevant mutations, P21L and W30R, on the activity of DHFR was evaluated via measurement of Michaelis-Menten and inhibitory kinetics, and structural characterization was undertaken by native mass spectrometry with ultraviolet photodissociation (UVPD). Compared to WT-DHFR, both P21L and W30R mutants produced less stable complexes with TMP in the presence of co-factor NADPH as evidenced by the relative abundances of complexes observed in ESI mass spectra. Moreover, based on variations in the fragmentation patterns obtained by UVPD mass spectrometry of binary and ternary DHFR complexes, notable structural changes were localized to the substrate binding pocket for W30R and to the M20 loop region as well as the C-terminal portion containing the essential G-H functional loop for the P21L mutant. The results suggest that the mutations confer resistance through distinctive mechanisms. A novel propargyl-linked antifolate compound 1038 was shown to be a reasonably effective inhibitor of the P21L mutant.
 ...
PMID:Characterization of trimethoprim resistant E. coli dihydrofolate reductase mutants by mass spectrometry and inhibition by propargyl-linked antifolates. 2996 75