3D Biomolecular Interaction Analysis

Small Molecule Screen

Small Molecule Screening with MIK-MS

SKi Pro, for the first time, allows users to screen small molecules for binding with label-free interaction analysis equipment coupled to mass spectrometry. While small molecule binding may be readily detected in refractive index mode, using MIK-MS allows multiple weak small molecule interactions to be characterized at once. More information and details of these techniques are available on our application notes.

Mass-Spec Sensorgram

Figure 1 Sampling scheme shown in (A), while the resulting mass spec data is shown in (B).

Mass Spec Sensorgram

A mass spectrometer may be used as a direct replacement for the refractive index detection scheme deployed by SKi Pro. In the small molecule binding context, instead of looking for an, almost always, <1% change in the refractive index signal, one is looking for a large change in the mass spectrometer signal.

Shown in Figure 1A is the fraction collection scheme for generating a mass spec sensorgram for the furosemide-carbonic anhydrase system. 30 second fractions of SKi Pro eluent are collected from both the sample and reference channels simultaneously. These are then analyzed by LC-MS and the signal changed to moles based on the previously obtained calibration curves for the analyte as shown in B.

This data show how a 0.5% change expected from refractive index detection, can be changed into a 25% change in mass spectrometric detection. This ends up being double advantageous in the context of MIK-MS for small molecules of pharmaceutically relevant compounds as those tend to be readily ionizable.

The fully parallel design of the fluidics of system coupled with best in class capture capacity mean these experiments are only possible with SKi Pro. No other existing system will allow time resolved MIK-MS data of the sort shown here.

Small Molecule Screening

SKi sensors have so much capture capacity it is possible to take real time resolved MIK-MS data using them. This fact also allows one to screen multiple compounds at the same time.

Figure 2 shows carbonic anhydrase II immobilized to the chip and seven separate sulfonamides are interacted at the same time (sulpiride, furosemide, dansylamide, benzene-disulfonamide, acetazolamide, sulfanilamide, benzenesulfonamide). The label free binding signal shows association, but it is not possible to tell which compounds contribute to the association. Bins are therefore taken during dissociation and the eluent is routed into an LC-MS setup. The changes in intensity of the first (blue) and second (red) bins can be seen, giving an immediate glimpse into the off-rate for multiple analytes at once.

The ratio of the mass spec measured intensities can be plotted against the known off rates of these compounds to show that the MS signal allows for rapid detection of binding and ranking of binders by off rate.

Using screening techniques such as these, customers have been able to screen for binders with affinities as low as KD=600 ┬ÁM, measuring up to ten analytes every 10 minutes.

Mass-Spec Sensorgram

Figure 2 (top) Label free binding signal for seven simultaneous analytes. (left) Sample mass spec signals for two of them. (right) Correlation of mass spec and label free binding signals.