(1307-D) High-throughput Affinity Selection Mass Spectrometry Using a Novel Two Dimensional RapidFire TOF Platform
Tuesday, February 6, 2024
2:00 PM – 3:00 PM EST
Location: Exhibit Halls AB
Abstract: Affinity Selection Mass Spectrometry (ASMS) is a solution-phase and label-free technique that enables the binding between small molecules and macro-molecule targets to be determined and measured. In one form of ASMS assay, the target and any bound small molecules are physically separated from unbound small molecules via size exclusion chromatography (SEC) under native conditions. The target fraction from this first dimension is automatically applied to a reverse-phase (RP) second dimension, which separates the binders from the target and delivers them to the mass spectrometer for measurement. This two-dimensional (2D) liquid chromatography (LC) mass spectrometry (MS) method been used extensively to identify and measure small molecule binding to proteins and oligonucleotide targets alike. Recently, ASMS has proven particularly useful in researching molecular glues, PROTAC degraders, and small molecules that can disrupt protein-protein interactions.
In this work, the modification and application of a RapidFire (RF) MS system to execute high-throughput ASMS is described. By introducing a SEC column upstream of the inherent RP cartridge, and leveraging the ability of RapidFire to switch between three solvents instantaneously, the 2D RF MS system was able to execute dissociation constant (KD) measurements at a sustained cycle time of ~1 minute per sample, representing a significant increase in throughput from what is typically achieved with 2D LC MS. Dissociation constant experiments for multiple established small molecule target pairs were analyzed and results between 2D RF MS and 2D LC MS matched. Two-dimensional RF, therefore, can provide high quality KD data on hits from primary screens much faster than traditional methods and facilitate hit follow-up and drug discovery, in general.