What Is Affinity Selection-Mass Spectrometry?

Introduction

Affinity selection-mass spectrometry (AS-MS), also referred to as Automated Ligand Identification System (ALIS), is a high-throughput technique designed to screen a drug target for small molecule, non-covalent binders. The drug target is typically proteinaceous but can also be other large biomolecules such as DNA, RNA, or a protein complex.  Candidate small molecule(s) are incubated with the target for a defined time period, followed by a filtration step to separate [target + bound molecule(s)] from [unbound material].

Finally, the bound molecules are liberated from the target by organic solvents using liquid chromatography coupled to high-resolution mass spectrometry.  The mass spectral data are processed to determine which candidate molecules are present.  Any molecules found represent potential hit matter that has affinity for the target and therefore warrant further investigation.

Many variations of the AS-MS experimental workflow are in use today including high throughput screening and hit validation workflows. These workflows typically differ in the number of candidate small molecules per reaction mixture (i.e. well).

Compression Ratios

The compression ratio refers to the number of candidate small molecules per reaction mixture.

High throughput screening workflows can have compression ratios ranging from 10 – 3,000 molecules per well. Organizations with large compound libraries may use AS-MS to rapidly screen their entire library, or specific focused libraries against a particular biological target. Processing the resulting data has historically been a challenge for several reasons including the large volume of data and the lack of useful software to help with analysis.  

Hit validation workflows generally use small compression ratios (1 – 10 molecules per well) as compared to screening workflows. In addition, several different validation scenarios can be prepared. For example, a single compound can be prepared with the drug target, without the drug target, or with a competitor target. An injection from each scenario is made along with a blank, standard, or variation thereof, and the results from each sample are compared to determine the hit status of the compound(s) in the well.

Software Automation in AS-MS

Virscidian provides a software solution for AS-MS workflows that efficiently manages the volume of data, provides multiple layers of analysis and filtering to confirm the presence of compounds, and reduces both false negatives and false positives. The software works off-the-shelf for many scenarios but can be customized for a specific workflow and customer needs. Due to several recent successes and the high-throughput nature of this experimental technique, AS-MS coupled with Virscidian’s software solution has become a powerful technique in small molecule drug discovery.