Frontal affinity chromatography for the screening of mixtures.
A protein stationary phase for frontal affinity chromatography was prepared, containing biotinylated beta-galactosidase immobilized to controlled pore glass beads via covalently bonded streptavidin. Single microaffinity columns of approximately 30 pmol of active beta-galactosidase were prepared from this material and characterized with a known ligand by frontal analysis. These columns were used to measure the specific interactions between the bound beta-galactosidase and a library of modified beta-galactopyranosides using electrospray mass spectrometry as the means of detection. The library contained 89 entries, each representing 4 diastereomers for a total of 356 library members. A single entry was analysed revealing differential activity among the 4 isomers. The library was grouped into 10 mixtures of 24-40 members each with each mixture infused under frontal chromatographic conditions. This deconvolution procedure led to the identification of 34 entries containing isomers with K(d) values better than 10 microM. A method based on a displacement principle was implemented as a rapid prescreen which served as the basis for a parallel column high throughput screening assay.
MDS Proteomics, 140-3553 31st Street N.W., Calgary, Alberta, Canada, T2L 2K7.
N W C
NW
Chan
D F
DF
Lewis
S
S
Hewko
O
O
Hindsgaul
D C
DC
Schriemer
D002846:Chromatography, Affinity / Q000379:methods*; D020650:Combinatorial Chemistry Techniques; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D005690:Galactose / Q000737:chemistry / Q000378:metabolism; D007536:Isomerism; D001616:beta-Galactosidase / Q000737:chemistry / Q000378:metabolism
D001616:beta-Galactosidase; D005690:Galactose