Frontal Affinity Chromatography for the Screening of Mixtures

Abstract

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.

Lectins: Proteins That Interpret the Sugar Code

“…FAC assays require that the buffer conditions for maximum activity and minimum interference with the desorption/ionization process at the ESI interface be optimized. Recently, a FAC/MS study of a library of 356 compounds targeted to ␤-galactosidase revealed 34 substances with dissociation constant values lower than 10 µM (14). However, not all of the tested compounds could be detected by ESI-MS; therefore, other ionization modes such as atmospheric pressure chemical ionization or MALDI may be needed to complement future assays.…”

Peer Reviewed: Biosensor Alternative: Frontal Affinity Chromatography

“…The concentration of the weaker ligand increases transiently above its infusion level; this is caused by the action of the slower-eluting stronger ligand. When the stronger ligand enters a region through which the weaker ligand has passed, it re-establishes the local equilibrium and displaces the weaker ligand (14,15). This wave, or roll-up, is a unique and useful feature that can be used to extract information from very complex mixtures.…”

“…FAC/MS is ideally suited for this purpose and can be applied to pooled combinatorial libraries or even natural product extracts. In a model system designed for a library with a high "hit rate", we screened a 356-member library of modified ␤-galactopyranosides with a FAC column containing immobilized ␤-galactosidase enzyme (15). Mixtures of 24 -40 compounds were infused, and the effluent was detected by online MS.…”

Frontal Affinity Chromatography with MS Detection of EphB2 Tyrosine Kinase Receptor. 2. Identification of Small-Molecule Inhibitors via Coupling with Virtual Screening

“…FAC-MS can also be used in a more rigorous mode for determining equilibrium binding constants, K d , of individual compounds. 8 The principles of the FAC-MS screening technique have been described elsewhere [5][6][7][8][9] but will be briefly summarized. It is based on the continuous infusion of small molecules over a protein target immobilized onto a solid support column followed by MS detection.…”