Affinity Selection Mass Spectrometry (AS-MS) is an advanced analytical technique used to identify and characterize molecular interactions. AS-MS helps scientists understand how different substances interact at a molecular level. It is a high-throughput technique that screens drug targets for small molecule binders.
The “Affinity Selection” Part
Affinity selection describes the natural tendency of molecules to bind specifically to one another. This process is about isolating or enriching molecules that interact with a target.
The method leverages these binding principles to separate desired molecules from a complex mixture. By introducing a target molecule, scientists can “select” or capture only those other molecules that form a stable interaction with it. This initial selection step filters out non-binding molecules, greatly simplifying subsequent analysis. The technique is not limited to proteins; it can also be applied to other biomolecules like DNA or RNA.
The “Mass Spectrometry” Part
Mass spectrometry (MS) is an analytical technique that measures the mass-to-charge ratio of ions. This measurement allows scientists to identify different molecules based on their unique “molecular fingerprint.” The process begins by converting neutral molecules into charged ions, either by gaining or losing an electron.
Once ionized, these charged particles are then separated in a vacuum based on how they move through electric or magnetic fields. Finally, a detector records the arrival of these separated ions, generating a mass spectrum that plots ion abundance against their mass-to-charge ratios. This spectrum provides information about the molecular mass and structural information.
Putting It Together: How AS-MS Works
The AS-MS process begins by exposing an “affinity bait,” such as a protein target, to a diverse collection of potential binding partners, often referred to as a library. This library can contain thousands of small molecules or natural product extracts. The target and potential binders are incubated together, allowing time for specific molecular interactions to occur.
After incubation, non-binding molecules are washed away, leaving behind only the complexes where a library molecule has “selected” and bound to the affinity bait. Various separation techniques are used for this step, including pulsed ultrafiltration (PUF), size exclusion chromatography (SEC), or magnetic microbead affinity selection screening (MagMASS). In MagMASS, the bait is immobilized on magnetic beads to separate bound from unbound molecules.
The selected molecules are then released from the bait using methods like organic solvents or pH changes. These released molecules are subsequently introduced into a high-resolution mass spectrometer. The mass spectrometer identifies the precise mass-to-charge ratio of each bound molecule, allowing for its identification and quantification. This integrated workflow provides a direct and rapid method for discovering lead compounds in libraries by isolating and identifying active components.
Real-World Applications
AS-MS plays a significant role in drug discovery, particularly in identifying new drug candidates and understanding how drugs interact with their biological targets. It is used to screen large compound libraries, evaluating their affinity to various biological targets like proteins, enzymes, DNA, or RNA. For example, AS-MS has facilitated the discovery of “molecular glues,” a class of small molecules that stabilize interactions between proteins, leading to targeted protein degradation, which is a novel approach in cancer therapy. One such molecular glue, HQ461, was identified to promote the interaction between CDK12 and an E3 ubiquitin ligase complex, leading to the degradation of cyclin K and inducing tumor cell death.
Beyond drug discovery, AS-MS is also applied in biomarker discovery for diseases and in the development of diagnostics. It can identify non-covalent binding molecules for challenging protein and oligonucleotide targets, especially those that do not show functional activity in traditional assays. The technique has been used to prospect for ligands in natural product libraries, identifying bioactive molecules from complex botanical, fungal, and microbial extracts. For instance, it helped identify cannabigerolic acid (CBGA) and cannabidiolic acid (CBDA) as ligands with high affinity for the SARS-CoV-2 spike protein S1 subunit, which were then shown to block the infection of human epithelial cells by a pseudovirus.
Why AS-MS Is a Powerful Tool
AS-MS offers several advantages that make it a powerful analytical tool in various scientific disciplines. It enables the rapid screening of large libraries of molecules, allowing for high-throughput analysis of thousands of compounds per well in screening workflows. This capability significantly accelerates the identification of potential binders compared to traditional methods that test one compound at a time. The technique is also highly sensitive, capable of detecting weak or transient molecular interactions that might be missed by other screening methods.
Furthermore, AS-MS does not require prior labeling of the molecules, such as with radiolabels or fluorescent dyes, which simplifies experimental setup and avoids potential interference from labels. It can identify unknown binders directly by their exact mass, providing elemental composition and structural information. This label-free and unbiased approach allows for the discovery of diverse ligands, including those that bind at the active site (orthosteric) or at other sites on the target (allosteric), which is a significant advantage in exploring new therapeutic possibilities.