April 19, 2021

High-throughput screening remains one of the most powerful, unbiased approaches in drug discovery for revealing novel small molecule modulators of biochemical and cellular activities. However, not all therapeutic targets exhibit functional activity and are therefore not amenable to traditional biochemical assays. Affinity selection mass spectrometry (ASMS) has emerged as an attractive screening strategy for challenging protein and oligonucleotide targets, particularly those that do not exhibit functional activity. The emergence of targeted protein degradation through PROTACs, for example, has motivated the need for rapid screening assays that identify non-covalent binding molecules to initiate development. The general ASMS strategy encompasses incubating the target with a single or pool of small molecules, isolating the target-small molecule complex, and then detecting the binding molecule using mass spectrometry. This workflow has been applied with some variations using distinct methodologies and MS instrumentation. Understanding the advantages and limitations of these approaches is critical for selecting which technique is suitable for your target and gives the highest likelihood for achieving your drug discovery goals.

Here are three important aspects to consider when selecting an ASMS methodology for your next drug discovery screening project:

1. Small molecule library. When a screening strategy relies on detecting the mass ID of the molecule of interest, it is imperative that the compounds in the library are sufficiently pure. Impurities such as degradation products can result in false negatives if the anticipated mass is not present, but also false positives, if the degradation product overlaps with the mass ID of another molecule in the well. Eliminating PAINS compounds is an additional filter to ensure the quality of the library.

Another common strategy is to pool small molecule libraries to increase throughput. This approach is common with slower workflows, such as those that rely on liquid chromatography to maintain a throughput suitable for large screening campaigns. However, pooling hundreds or thousands of compounds in a single reaction can introduce artifacts. It is important to consider how large compound pools increase the probability of having competitive binders present in a single reaction and how compound behavior—including solubility and aggregation—is impacted by the pooling approach.

Notably, faster MS approaches, including those that rely on MALDI MS, are able to maintain high-throughput capacities with pools of < 10 compounds. A balance of throughput and compound pooling must be achieved for optimal success.

2. ASMS methodology. ASMS techniques have evolved since the initial description over 15 years ago and several distinct approaches have been reported. Each approach varies in the experimental design, the throughput, the reagent requirements, and the mass spectrometer instrument. Below is a breakdown of notable aspects of three popular approaches:

Automated ligand identification system (ALIS)

  • Relies on multiple chromatography steps to first isolate the small molecule-target complex, followed by a second separation step to dissociate the complex prior to MS detection using an ESI source
  • Pools hundreds to thousands of compounds in a single reaction to increase throughput
  • Compounds may be susceptible to fragmentation, and dedicated software packages are important for deconvolution, data analysis, and hit identification
  • Target screening concentration requirements are significant (~10 mM)


  • Incorporates size exclusion chromatography resin in a 384-filter plate assay that enables purification of 384 samples simultaneously
  • Utilizes a multistep process that involves filtration and elution, followed by detection using high-throughput MALDI MS
  • The high-throughput capacity allows the screening of smaller pools or individual compounds
  • Target screening concentration requirements are significant (~10 mM), and quality control reagents must be included to measure success of filtering and elution, along with internal standards to identify hits


  • Innovations with self-assembled monolayers have enabled the immobilization of small-molecule target complexes in a 384-plate format followed by detection of bound compounds by MALDI MS, allowing the screening of diverse targets
  • The monolayer, functionalized to minimize nonspecific protein adsorption, also serves as an internal standard to compare the binding of candidate compounds
  • The high-throughput capacity of MALDI is amenable to small compound pool sizes (<10 compounds)
  • Target screening concentration requirements are up to 50-fold less than other approaches

Understanding how these methodologies and their associated workflows may impact your drug discovery goals is important. For example, compounds with weaker affinities (> 50 mM) may not be detected, or compounds with rapid off-rates may be lost during separation or purification steps. Alternatively, the ability to screen over a half-million compounds in a matter of days and rank order compounds will rapidly highlight the most promising hits for further development. Of course, speed is only one aspect; the data quality, discussed next, is paramount.

3. Data quality. The most important aspect of any screen is obtaining reliable results. The first step in gaining confidence in your hits is by confirming them in follow-up ASMS experiments with replicates and ultimately in dose response formats to rank order the lead compounds. Once hits are identified and confirmed, the next step is validation through orthogonal assays.

Developing orthogonal assays may be challenging for nonfunctional targets that cannot be interrogated with traditional biochemical assays. Importantly, some binders may not exhibit functional activity if they do not impact the catalytic site of a given target. Binding assays offer a more promising validation strategy. While high-throughput assays such as FRET and radioactivity are available in a competition assay format, they require the synthesis of a labeled reporter, and optical readouts are prone to interference from library compounds. A common strategy is therefore to shift toward lower throughput, quantitative biophysical techniques such as:

  • Surface plasmon resonance (SPR)
  • Differential scanning fluorimetry (DSF) (along with other thermal shift assays)
  • Nuclear magnetic resonance (NMR)

Like ASMS methodologies, each of these techniques differs in its capabilities. Understanding the advantages and limits of detection is important and plays a critical role in validating the ASMS data. As with any drug discovery program, carefully considering all advantages and identifying potential challenges will help you maximize your success.

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