On June 03, 2025, a groundbreaking study titled "Native mass spectrometry facilitates hit validation in DNA-encoded library technology" was published in Angewandte Chemie International Edition by Bittner and colleagues. This research introduces native mass spectrometry (MS) as a transformative solution for addressing critical bottlenecks in DEL hit validation, demonstrating for the first time a methodology that enables rapid, label-free analysis of protein-on-DNA ligand complexes. By preserving noncovalent interactions in the gas phase through a sophisticated workflow involving buffer exchange and soft ionization techniques, the team achieved reliable determination of dissociation constants (Kd) across diverse protein targets, from human carbonic anhydrase to kinase systems. The study highlights the method's ability to validate binders directly from unpurified synthesis mixtures, offering structural insights for medicinal chemistry while complementing advanced library technologies like CD BioGlyco's DNA-encoded Glycan Library (DEGL) Services. These breakthroughs pave the way for more efficient drug discovery workflows, particularly in glycan-focused applications where CD BioGlyco's specialized expertise provides a synergistic advantage.

The DEL Validation Challenge

DEL technology utilizes collections of millions to billions of structures, each identifiable by a covalently linked DNA barcode. While affinity-based selections can test enormous compound libraries against target proteins, the subsequent validation of individual hits has traditionally been problematic. Conventional methods such as isothermal titration calorimetry (ITC), fluorescence polarization (FP), and surface plasmon resonance (SPR) often consume substantial amounts of target protein or synthesized ligand, making them suboptimal for high-throughput applications. Furthermore, these methods work best for high-affinity ligands, while many DEL-derived hits exhibit moderate to low affinity when first discovered. This validation bottleneck becomes particularly challenging when working with complex targets like glycans, where CD BioGlyco's specialized expertise in carbohydrate research provides distinct advantages.

Native MS Methodology Breakthrough

The research team developed a sophisticated native MS workflow that preserves noncovalent interactions in the gas phase, enabling direct analysis of protein-on-DNA ligand complexes without labeling or immobilization. The experimental setup involves buffer-exchanging both protein and on-DNA ligand into volatile ammonium acetate solution, followed by nano-electrospray ionization under soft tune conditions. This approach maintains the native folded state of proteins while monitoring binding events through intensity changes in protein-ligand complex peaks.

Fig. 1. Determination of dissociation constants (Kd) for on-DNA ligands against protein targets by native MS. (Bittner, et al., 2025)

The methodology was rigorously validated using human carbonic anhydrase II (hCAII) as a model system, demonstrating reliable determination of dissociation constants (Kd) for on-DNA ligands across a wide affinity range (nanomolar to micromolar). The researchers successfully extended their approach to more complex systems, including bromodomain-containing protein 4 (BRD4-BD1) and Bruton's tyrosine kinase (BTK), confirming the general applicability of their method. This technical advancement aligns perfectly with CD BioGlyco's focus on developing robust analytical methods for complex biomolecular interactions.

Unprecedented Advantages in Hit Validation

• Rapid Verification Without Purification

One of the most significant advancements demonstrated in this study is the ability to validate binding events using unpurified on-DNA synthesis mixtures. Traditional validation requires HPLC purification of potential hits, which becomes impractical when dealing with numerous candidates. The native MS approach successfully identified true binders from crude mixtures containing precursors and side products, dramatically accelerating the validation timeline.

Fig.2 Direct analysis of on-DNA ligand binding to BTK from an unpurified mixture. (Bittner, et al., 2025)

• Structural Insights for Medicinal Chemistry

Beyond simple binding confirmation, native MS provides valuable information about which substructures or synthons contribute most significantly to protein binding. The researchers demonstrated this capability using a BTK ligand (BTK1) that hydrolyzed under acidic conditions. Remarkably, the MS analysis revealed binding not only of the full ligand but also of hydrolyzed precursors and individual building blocks, offering medicinal chemists crucial insights for lead optimization.

• Compatibility with Different DNA Conjugation Systems

The study comprehensively evaluated both single-stranded and double-stranded DNA conjugation systems commonly used in DEL technology. While both systems were compatible with the native MS approach, interesting differences in binding affinities were observed, providing new insights into how DNA linkage strategies influence ligand presentation and binding. This finding has particular relevance for glycan-based library design, where CD BioGlyco's expertise in DNA-compatible glycan chemistry offers unique advantages.

Integration with Advanced DEGL Technology

The groundbreaking methodology described in this research aligns perfectly with the innovative services offered by CD BioGlyco in the field of DEGL technology. As pioneers in carbohydrate research and DEL applications, we provide comprehensive solutions that complement and enhance the native MS validation approach.

Our expertise in DEGL design and optimization, DNA-compatible reaction development, and library construction creates an ideal foundation for implementing advanced validation strategies like native MS. Their high-throughput screening capabilities, combined with next-generation sequencing and sophisticated data analysis, provide a complete workflow that integrates seamlessly with the validation approach demonstrated in this research.

For researchers looking to leverage these cutting-edge technologies, we offer:

Future Perspectives and Applications

The native MS methodology represents a significant leap forward in DEL hit validation, with potential applications extending beyond the examples demonstrated in the current study. The researchers note that while their approach might face challenges with heavily glycosylated protein targets, it can still demonstrate binding events in these cases. Future developments may include high-throughput implementations using arrayed 96-well emitters and automated setups, potentially reducing measurement times for full titration series to less than an hour.

The observation that different DNA conjugation systems affect binding affinities opens new avenues for optimizing library design. As the researchers indicate, their findings "can also be employed to further improve DEL technology by optimizing the compound display in DNA-encoded libraries, since it has been shown that the linker design utilized to present the combinatorially synthesized library members can have a significant impact on binding."

Conclusion

The integration of native mass spectrometry into DEL hit validation workflows marks a transformative advancement in drug discovery technology. The methodology developed by Bittner, Gloger, and colleagues addresses critical bottlenecks in hit validation while providing unprecedented insights into binding mechanisms. This approach, when combined with sophisticated library technologies like those offered by CD BioGlyco, creates a powerful platform for accelerating drug discovery across multiple target classes.

For researchers interested in implementing these cutting-edge technologies, CD BioGlyco provides the expertise and infrastructure necessary to leverage these advancements in both glycan-focused and small-molecule drug discovery programs. The synergy between advanced validation methodologies and sophisticated library technologies represents the future of efficient and effective drug discovery.

To explore how these advanced validation technologies can enhance your drug discovery programs, visit CD BioGlyco's DEGL services or contact our expert team for customized solutions.